Follistatin-3

ABSTRACT

The present invention relates to a novel follistatin-3 protein which is a member of the family of inhibin-related proteins. In particular, isolated nucleic acid molecules are provided encoding the human follistatin-3 protein. Follistatin-3 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of follistatin-3 activity. Also provided are diagnostic methods for detecting reproductive system-related disorders and disorders of the regulation of cell growth and differentiation and therapeutic methods for treating reproductive system-related disorders and disorders of the regulation of cell growth and differentiation.

[0001] This application is a Divisional of U.S. application Ser. No.09/617,804, filed Jul. 14, 2000, which claims benefit under 35 U.S.C.§119(e) of the filing date of U.S. Provisional Application Serial No.60/144,088, filed Jul. 16, 1999, and also claims benefit of priorityunder 35 U.S.C. §120 of the filing date of copending U.S. applicationSer. No. 09/141,027 filed Aug. 27, 1998, which in turn claims benefit ofpriority under 35 U.S.C. §119(e) to U.S. Provisional Application SerialNo. 60/056,248, filed Aug. 29, 1997; U.S. application Ser. No.09/617,804 also claims benefit of priority under 35 U.S.C. §120 of thefiling date of copending PCT Application Serial No. PCT/US98/17710,filed Aug. 27, 1998; each of which is hereby incorporated by referencein their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to a novel human gene encoding apolypeptide which is a member of the family of inhibin-related proteins.More specifically, isolated nucleic acid molecules are provided encodinga human polypeptide named follistatin-3. Follistatin-3 polypeptides arealso provided, as are vectors, host cells and recombinant methods forproducing the same. Also provided are diagnostic methods for detectingdisorders related to the reproductive system, and therapeutic methodsfor treating such disorders. The invention further relates to screeningmethods for identifying agonists and antagonists of follistatin-3activity.

BACKGROUND OF THE INVENTION

[0003] The family of inhibin-related proteins currently consists of atleast four groups of members: inhibins, activins, and two splicevariants of follistatin-1 (315 and 288 amino acids). Inhibins andactivins are members of the transforming growth factor (TGF)-betasuperfamily and function with opposing actions in a variety ofcapacities in paracrine and autocrine regulation of both reproductiveand nonreproductive organs including the liver, kidney, adrenal glands,bone marrow, placenta, anterior pituitary, and brain (Ying, S. Y., etal., Proc. Soc. Exp. Biol. Med. 214:114-122 (1997); Mather, J. P., etal., Proc. Soc. Exp. Biol. Med. 215:209-222 (1997)). Although thefollistatins are not closely related to the TGF-beta family, they stillplay a major role in the follical stimulating hormone (FSH) syntheticpathway by increasing estradiol production and by functioning directlyas high affinity activin-binding proteins. Inhibins, activins, andfollistatin-1 were all initially identified as regulators of pituitaryFSH secretion, but have more recently been further characterized tofunction as growth factors, embryo modulators, and immune factors(Petraglia, F. Placenta 18:3-8 (1997)). In addition, each of thesefactors is involved with the regulation of gonadotropin biosynthesis andsecretion, ovarian and placental steroidogenesis, and oocyte andspermatogonial maturation (Halvorson, L. M. and DeCherney, A. H. Fertil.Steril. 65:459-469 (1996)).

[0004] FSH is a vital component of the regulatory cascade governingdevelopment of human oocytes. Primary oocytes in newborns are arrestedin the prophase stage of Meiosis I and are surrounded by a 1-2 cellthick layer of follicle cells constituting a structure termed theprimordial follicle. In concert with other factors, stimulation of theprimordial follicle with FSH initiates its progression to the morecomplex structures designated the developing and antral follicles (Ueno,N., et al., Proc. Natl. Acad. Sci. USA 84:8282-8286 (1987); Robertson,D. M., et al., Biochem. Biophys. Res. Comm. 149:744-749 (1987)). Theantral follicle consists of an enlarged oocyte surrounded by anincreased number of follicle cells, a zona pellucida, cortical granules,and a fluid-filled cavity termed the antrum. It is in this state thatthousands of developing oocytes are maintained until puberty. Each monthfollowing this point, a surge in the local concentration of severaladditional hormones and other factors, primarily leuteinizing hormone(LH), stimulates accelerates the growth of roughly 15-20 of thedeveloping follicles in the ovary. Only one of these structures willultimately complete the developmental progression of its enclosed oocyteto the metaphase stage of Meiosis II. The single stimulated folliclewill then continue to enlarge until it bursts at the surface of theovary and releases the oocyte, still surrounded with a coating offollicle cells, for potential fertilization (Bornslaeger, E. A., et al.,Dev. Biol. 114:453-462 (1986); Masui, Y. and Clarke, H. J. Int. Rev.Cytol. 57:185-282 (1979); Richards, J. S. Recent Prog. Horm. Res.35:343-373 (1979)).

[0005] Follistatin also plays a central role in the above-describedprocess of follicle development. Follistatin binds stoichiometrically toactivins and, as a result, inhibits the activin-induced augmentation ofFSH-release from cultured pituitary cells (Kogawa, K., et al.,Endocrinology 128:1434-1440 (1991)). Further evidencing a feedbackmechanism, cultured granulosa cells produce and secrete follistatin inresponse to treatment with FSH (Saito, S., et al., Biochem. Biophys.Res. Comm. 176:413-422 (1991); Klein, R., et al., Endocrinology128:1048-1056 (1991)). Furthermore, it has been determined bysynthesizing the results of a number of studies, that follistatin,activin, FSH, LH, and other factors function in concert in a variety ofinterrelated mechanisms to regulate many developmental processes,including the development of follicles. For example, in the presence ofFSH, activin can augment both LH receptor expression and progesteroneproduction by rat granulosa cells (Sugino, H., et al, Biochem. Biophys.Res. Comm. 153:281-288 (1988)). In addition, activin can significantlyenhance the ability of granulosa cells to express FSH receptor andproduce inhibin even in the absence of FSH (Nakamura, T., et al.,Biochim. Biophys. Acta 1135:103-109 (1992); Sugino, H., et al., supra;Hasegawa, Y., et al., Biochem. Biophys. Res. Comm. 156:668-674 (1988)).These and other studies provide support for the idea that follistatinand activin play important roles in the regulation of granulosa cellulardifferentiation.

[0006] In addition to the many well-characterized effects whichfollistatin, activin, and inhibin elicit on the regulation of variousdevelopmental processes in the reproductive system, a large number ofstudies have more recently begun to define regulatory roles for thesemolecules in a variety of other tissues and systems. For example, duringearly embryonic development in Xenopus laevis, the action of activin Ain developing targets of ciliary ganglion neurons is regulated bylocalized expression of follistatin (Hemmati-Brivanlou, A. and Melton,D. A. Nature 359:609-614 (1992); Hemmati-Brivanlou, A. and Melton, D. A.Cell 77:273-281 (1994)). In addition, overexpression of follistatinleads to induction of neural tissue (Hemmati-Brivanlou, A., et al., Cell77:283-295 (1994)). In the mouse, follistatin mRNA is first detected onembryonic day 5.5 in the deciduum, and, subsequently, in the developinghindbrain, somites, vibrissae, teeth, epidermis, and muscle (van denEihnden-van Raaij, A. J. M., et al., Dev. Biol. 154:356-365 (1992);Albano, R. M., et al., Development 120:803-813 (1994); Feijen, A., etal., Development 120:3621-3637 (1994)). Evidence of the relativeimportance of such a varied expression of follistatin is provided byMatzuk and colleagues (Nature 374:360-363 (1995)) who demonstrate thatfollistatin-deficient mice are retarded in their growth, have decreasedmass of the diaphragm and intercostal muscles, shiny taut skin, skeletaldefects of the hard palate and the thirteenth pair of fibs, theirwhisker and tooth development is abnormal, they fail to breathe, and diewithin hours of birth. Since the defects in mice deficient infollistatin are far more widespread than in mice deficient in activin,Matzuk and coworkers (supra) suggest that follistatin may modulate thecell growth and differentiation regulatory actions of additional membersof the TGF-beta superfamily.

[0007] Thus, there is a need for polypeptides that function asregulators of reproductive development, embryonic development, and cellgrowth and differentiation since disturbances of such regulation may beinvolved in disorders relating to reproduction and the regulation ofcell growth and differentiation. Therefore, there is a need foridentification and characterization of such human polypeptides which canplay a role in detecting, preventing, ameliorating or correcting suchdisorders.

SUMMARY OF THE INVENTION

[0008] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of thefollistatin-3 polypeptide having the complete amino acid sequence shownin SEQ ID NO:2 or the complete amino acid sequence encoded by the cDNAclone deposited as plasmid DNA as ATCC® Deposit Number 209199 on Aug. 8,1997. The nucleotide sequence determined by sequencing the depositedfollistatin-3 clone, which is shown in FIGS. 1A, 1B, and 1C (SEQ IDNO:1), contains an open reading frame encoding a complete polypeptide of263 amino acid residues, including an initiation codon encoding anN-terminal methionine at nucleotide positions 19-21, and a predictedmolecular weight of about 27.7 kDa. Nucleic acid molecules of theinvention include those encoding the complete amino acid sequenceexcepting the N-terminal methionine shown in SEQ ID NO:2, or thecomplete amino acid sequence excepting the N-terminal methionine encodedby the cDNA clone in ATCC® Deposit Number 209199, which molecules alsocan encode additional amino acids fused to the N-terminus of thefollistatin-3 amino acid sequence.

[0009] The encoded polypeptide has a predicted leader sequence of 26amino acids underlined in FIG. 1A; and the amino acid sequence of thepredicted mature follistatin-3 protein is also shown in FIG. 1A, asamino acid residues 27-263 and as residues 1-237 in SEQ ID NO:2.

[0010] Thus, one aspect of the invention provides an isolated nucleicacid molecule comprising a polynucleotide comprising a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence encoding the follistatin-3 polypeptide having the completeamino acid sequence in SEQ ID NO:2 (i.e., positions −26 to 237 of SEQ IDNO:2); (b) a nucleotide sequence encoding the follistatin-3 polypeptidehaving the complete amino acid sequence in SEQ ID NO:2 excepting theN-terminal methionine (i.e., positions −25 to 237 of SEQ ID NO:2); (c) anucleotide sequence encoding the predicted mature follistatin-3polypeptide having the amino acid sequence at positions 1 to 237 in SEQID NO:2; (d) a nucleotide sequence encoding the follistatin-3polypeptide having the complete amino acid sequence encoded by the cDNAclone contained in ATCC® Deposit No. 209199; (e) a nucleotide sequenceencoding the follistatin-3 polypeptide having the complete amino acidsequence excepting the N-terminal methionine encoded by the cDNA clonecontained in ATCC® Deposit No. 209199; (f) a nucleotide sequenceencoding the mature follistatin-3 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC® Deposit No.209199; and (g) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e) or (f) above.

[0011] Further embodiments of the invention include isolated nucleicacid molecules that comprise a polynucleotide having a nucleotidesequence at least 90% identical, and more preferably at least 95%, 96%,97%, 98% or 99% identical, to any of the nucleotide sequences in (a),(b), (c), (d), (e), (f) or (g), above, or a polynucleotide whichhybridizes under stringent hybridization conditions to a polynucleotidein (a), (b), (c), (d), (e), (f) or (g), above. This polynucleotide whichhybridizes does not hybridize under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues.

[0012] An additional nucleic acid embodiment of the invention relates toan isolated nucleic acid molecule comprising a polynucleotide whichencodes the amino acid sequence of an epitope-bearing portion of afollistatin-3 polypeptide having an amino acid sequence in (a), (b),(c), (d), (e) or (f), above. A further embodiment of the inventionrelates to an isolated nucleic acid molecule comprising a polynucleotidewhich encodes the amino acid sequence of a follistatin-3 polypeptidehaving an amino acid sequence which contains at least one amino acidsubstitution, but not more than 50 amino acid substitutions, even morepreferably, not more than 40 amino acid substitutions, still morepreferably, not more than 30 amino acid substitutions, and still evenmore preferably, not more than 20 amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apolynucleotide which encodes the amino acid sequence of a follistatin-3polypeptide to have an amino acid sequence which contains not more than10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. Conservativesubstitutions are preferable.

[0013] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of follistatin-3 polypeptides or peptides by recombinanttechniques.

[0014] In accordance with a further aspect of the present invention,there is provided a process for producing such polypeptide byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing a follistatin-3 nucleic acidsequence, under conditions promoting expression of said protein andsubsequent recovery of said protein.

[0015] The invention further provides an isolated follistatin-3polypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence of the full-lengthfollistatin-3 polypeptide having the complete amino acid sequence shownin SEQ ID NO:2 (i.e., positions −26 to 237 of SEQ ID NO:2); (b) theamino acid sequence of the full-length follistatin-3 polypeptide havingthe complete amino acid sequence shown in SEQ ID NO:2 excepting theN-terminal methionine (i.e., positions −25 to 237 of SEQ ID NO:2); (c)the amino acid sequence of the predicted mature follistatin-3polypeptide having the amino acid sequence at positions 1 to 237 in SEQID NO:2; (d) the amino acid sequence of the full-length follistatin-3polypeptide having the complete amino acid sequence encoded by the cDNAclone contained in ATCC® Deposit No. 209199; (e) the amino acid sequenceof the full-length follistatin-3 polypeptide having the complete aminoacid sequence excepting the N-terminal methionine encoded by the cDNAclone contained in ATCC® Deposit No. 209199; and (f) the amino acidsequence of the mature follistatin-3 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC® Deposit No.209199. The polypeptides of the present invention also includepolypeptides having an amino acid sequence at least 80% identical, morepreferably at least 90% identical, and still more preferably 95%, 96%,97%, 98% or 99% identical to those described in (a), (b), (c), (d), (e)or (f) above, as well as polypeptides having an amino acid sequence withat least 90% similarity, and more preferably at least 95% similarity, tothose above.

[0016] An additional embodiment of this aspect of the invention relatesto a peptide or polypeptide which comprises the amino acid sequence ofan epitope-bearing portion of a follistatin-3 polypeptide having anamino acid sequence described in (a), (b), (c), (d), (e) or (f) above.Peptides or polypeptides having the amino acid sequence of anepitope-bearing portion of a follistatin-3 polypeptide of the inventioninclude portions of such polypeptides with at least six or seven,preferably at least nine, and more preferably at least about 30 aminoacids to about 50 amino acids, although epitope-bearing polypeptides ofany length up to and including the entire amino acid sequence of apolypeptide of the invention described above also are included in theinvention.

[0017] A further embodiment of the invention relates to a polypeptidewhich comprises the amino acid sequence of a follistatin-3 polypeptidehaving an amino acid sequence which contains at least one amino acidsubstitution, but not more than 50 amino acid substitutions, even morepreferably, not more than 40 amino acid substitutions, still morepreferably, not more than 30 amino acid substitutions, and still evenmore preferably, not more than 20 amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apeptide or polypeptide to have an amino acid sequence which comprisesthe amino acid sequence of a follistatin-3 polypeptide, which containsat least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 aminoacid substitutions. In specific embodiments, the number of additions,substitutions, and/or deletions in the amino acid sequence of FIGS. 1Aand 1B, FIGS. 2A and 2B, or fragments thereof (e.g., the mature formand/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50,10-50 or 50-150, conservative amino acid substitutions are preferable.

[0018] In another embodiment, the invention provides an isolatedantibody that binds specifically to a follistatin-3 polypeptide havingan amino acid sequence described in (a), (b), (c), (d), (e) or (f)above. The invention further provides methods for isolating antibodiesthat bind specifically to a follistatin-3 polypeptide having an aminoacid sequence as described herein. Such antibodies are usefuldiagnostically or therapeutically as described below.

[0019] The invention also provides for pharmaceutical compositionscomprising follistatin-3 polypeptides, particularly human follistatin-3polypeptides, which may be employed, for instance, to treat cancers andother cellular growth and differentiation disorders, as well asdisorders of the reproductive system. Methods of treating individuals inneed of follistatin-3 polypeptides are also provided.

[0020] The invention further provides compositions comprising afollistatin-3 polynucleotide or a follistatin-3 polypeptide foradministration to cells in vitro, to cells ex vivo and to cells in vivo,or to a multicellular organism. In certain particularly preferredembodiments of this aspect of the invention, the compositions comprise afollistatin-3 polynucleotide for expression of a follistatin-3polypeptide in a host organism for treatment of disease. Particularlypreferred in this regard is expression in a human patient for treatmentof a dysfunction associated with aberrant endogenous activity offollistatin-3.

[0021] The present invention also provides a screening method foridentifying compounds capable of enhancing or inhibiting a biologicalactivity of the follistatin-3 polypeptide, which involves contacting aligand which is inhibited by the follistatin-3 polypeptide with thecandidate compound in the presence of a follistatin-3 polypeptide,assaying receptor-binding activity of the ligand in the presence of thecandidate compound and of follistatin-3 polypeptide, and comparing theligand activity to a standard level of activity, the standard beingassayed when contact is made between the ligand itself in the presenceof the follistatin-3 polypeptide and the absence of the candidatecompound In this assay, an increase in ligand activity over the standardindicates that the candidate compound is an agonist of follistatin-3activity and a decrease in ligand activity compared to the standardindicates that the compound is an antagonist of follistatin-3 activity.

[0022] In another aspect, a screening assay for agonists and antagonistsis provided which involves determining the effect a candidate compoundhas on follistatin-3 binding to activin or an activin-like molecule. Inparticular, the method involves contacting the activin or anactivin-like molecule with a follistatin-3 polypeptide and a candidatecompound and determining whether follistatin-3 polypeptide binding tothe activin or an activin-like molecule is increased or decreased due tothe presence of the candidate compound. In this assay, an increase inbinding of follistatin-3 over the standard binding indicates that thecandidate compound is an agonist of follistatin-3 binding activity and adecrease in follistatin-3 binding compared to the standard indicatesthat the compound is an antagonist of follistatin-3 binding activity.

[0023] It has been discovered that follistatin-3 is expressed not onlyin Hodgkin's Lymphoma but also in synovial fibroblasts, gall bladder,resting and serum-induced smooth muscle, testes, Merkel cells, HELcells, hippocampus, TNF-alpha- and IFN-induced epithelial cells,keratinocyte, amygdala depression, HL-60 cells, hepatoma,progesterone-treated epidermal cells, endothelial cells, HSC172 cells,epitheloid sarcoma, activated T-cells, breast lymph node, pancreaticcarcinoma, fetal dura mater, fetal lung, epididymis, placenta, dendriticcells, rejected kidney, and uterine cancer. Therefore, nucleic acids ofthe invention are useful as hybridization probes for differentialidentification of the tissue(s) or cell type(s) present in a biologicalsample. Similarly, polypeptides and antibodies directed to thosepolypeptides are useful to provide immunological probes for differentialidentification of the tissue(s) or cell type(s). In addition, for anumber of disorders of the above tissues or cells, particularly of thereproductive system, or disorders of the regulation of cell growth anddifferentiation, significantly higher or lower levels of follistatin-3gene expression may be detected in certain tissues (e.g., cancerous andwounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovialfluid or spinal fluid) taken from an individual having such a disorder,relative to a “standard” follistatin-3 gene expression level, i.e., thefollistatin-3 expression level in healthy tissue from an individual nothaving the reproductive system or regulation of cell growth anddifferentiation disorder. Thus, the invention provides a diagnosticmethod useful during diagnosis of such a disorder, which involves: (a)assaying follistatin-3 gene expression level in cells or body fluid ofan individual; (b) comparing the follistatin-3 gene expression levelwith a standard follistatin-3 gene expression level, whereby an increaseor decrease in the assayed follistatin-3 gene expression level comparedto the standard expression level is indicative of disorder in thereproductive system or of a disorder of the regulation of cell growthand differentiation.

[0024] An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of follistatin-3activity in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an isolatedfollistatin-3 polypeptide of the invention or an agonist thereof.

[0025] A still further aspect of the invention is related to a methodfor treating an individual in need of a decreased level of follistatin-3activity in the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of anfollistatin-3 antagonist. Preferred antagonists for use in the presentinvention are follistatin-3-specific antibodies.

BRIEF DESCRIPTION OF THE FIGURES

[0026]FIGS. 1A, 1B, and 1C show the nucleotide sequence (SEQ ID NO:1)and deduced amino acid sequence (SEQ ID NO:2) of follistatin-3.

[0027] The predicted leader sequence of about 26 amino acids isunderlined. Note that the methionine residue at the beginning of theleader sequence in FIG. 1A is shown in position number (positive) 1,whereas the leader positions in the corresponding sequence of SEQ IDNO:2 are designated with negative position numbers. Thus, the leadersequence positions 1 to 26 in FIG. 1A correspond to positions −26 to −1in SEQ ID NO:2.

[0028] Two potential asparagine-linked glycosylation sites are marked inthe amino acid sequence of follistatin-3. The sites are asparagine-73and asparagine-215 in FIG. 1A (asparagine-47 and asparagine-179 in SEQID NO:2), and are with the bold pound symbol (#) above the nucleotidesequence coupled with a bolded one letter abbreviation for theasparagine (N) in the amino acid sequence in FIG. 1A; that is, theactual asparagine residues which are potentially glycosylated is boldedin FIG. 1A. The potential N-linked glycosylation sequences are found atthe following locations in the follistatin-3 amino acid sequence: N-73through H-76 (N-73, L-74, T-75, H-76) and N-215 through Y-218 (N-215,V-216, T-217, Y-218). A potential Protein Kinase C (PKC) phosphorylationsite is also marked in FIG. 1A with a bolded tyrosine symbol (T) in thefollistatin-3 amino acid sequence and an asterisk (*) above the firstnucleotide encoding that tyrosine residue in the follistatin-3nucleotide sequence. The potential PKC phosphorylation sequence is foundin the follistatin-3 amino acid sequence from residue T-141 throughresidue R-143 (T-141, Y-142, R-143). Potential Casein Kinase II (CK2)phosphorylation sites are also marked in FIG. 1A with a bolded tyrosineor serine symbol (T or S) in the follistatin-3 amino acid sequence andan asterisk (*) above the first nucleotide encoding the appropriatetyrosine or serine residue in the follistatin-3 nucleotide sequence.Potential CK2 phosphorylation sequences are found at the followinglocations in the follistatin-3 amino acid sequence: T-57 through E-60(T-57, R-58, A-59, E-60); T-141 through D-144 (T-141, Y-142, R-143,D-144); T-246 through E-249 (T-246, P-247, E-248, E-249); and S-255through E-258 (S-255, A-256, E-257, E-258). Ten potential myristylationsites are found in the follistatin-3 amino acid sequence shown in FIG.1A. Potential myristylation sites are marked in FIG. 1A with a doubleunderline delineating the amino acid residues representing eachpotential myristolation site in the follistatin-3 amino acid sequence.The potential myristolation sites are located in the following postionsin the follistatin-3 amino acid sequence: G-43 through C-48 (G-43, Q-44,E-45, A-46, T-47, C-48); G-65 through A-70 (G-65, N-66, I-67, D-68,T-69, A-70); G-78 through L-83 (G-78, N-79, K-80, I-81, N-82, L-83);G-88 through L-93 (G-88, L-89, V-90, H-91, C-92, L-93); G-136 throughT-141 (G-136, S-137, D-138, G-139, A-140, T-141); G-188 through V-193(G-188, S-189, A-190, H-191, C-192, V-193); G-207 through G-212 (G-207,Q-208, E-209, L-210, C-211, G-212); G-236 through G-241 (G-236, V-237,R-238, H-239, A-240, G-241); G-241 through T-246 (G-241, S-242, C-243,A-244, G-245, T-246); and G-252 through E-257 (G-252, G-253, E-254,S-255, A-256, E-257).

[0029]FIG. 2 shows the regions of identity between the amino acidsequences of the follistatin-3 protein and translation product of thehuman mRNA for follistatin-1 (SEQ ID NO:3), determined by the computerprogram Bestfit (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711) using the default parameters.

[0030]FIG. 3 shows an analysis of the follistatin-3 amino acid sequence.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability, as predicted using default parameters, are shown.

[0031] In the “Antigenic Index or Jameson-Wolf” graph, the positivepeaks indicate locations of the highly antigenic regions of thefollistatin-3 protein, i.e., regions from which epitope-bearing peptidesof the invention can be obtained. Non-limiting examples of antigenicpolypeptides or peptides that can be used to generatefollistatin-3-specific antibodies include: a polypeptide comprisingamino acid residues from about Lys-54 to about Asp-62, from about Val-91to about Leu-99, from about Lys-100 to about Gln-108, from about Cys-116to about Pro-124, from about Gln-140 to about Leu-148, from aboutTrp-156 to about Ser-164, from about Arg-170, to about Gln-181, fromabout Cys-212 to about Phe-224, from about Tyr-239, to about Thr-247,from about Pro-251, to about Met-259, and from about Asp-263, to aboutHis-271.

[0032] The data presented in FIG. 3 are also represented in tabular formin Table I. The columns are labeled with the headings “Res”, “Position”,and Roman Numerals I-XIV. The column headings refer to the followingfeatures of the amino acid sequence presented in FIG. 3 and Table I:“Res”: amino acid residue of SEQ ID NO:2 or FIG. 1A (which is theidentical sequence shown in SEQ If) NO:2, with the exception that theresidues are numbered 1-263 in FIG. 1A and −18 through 348 in SEQ IDNO:4); “Position”: position of the corresponding residue within SEQ IDNO:2 or FIGS. 2A and 2B (which is the identical sequence shown in SEQ IDNO:4, with the exception that the residues are numbered 1-366 in FIGS.2A and 2B and −18 through 348 in SEQ ID NO:4); I: Alpha,Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III: Beta,Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn,Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil,Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX:Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg;XI: Beta, Amphipathic Regions—Eisenberg; XII: FlexibleRegions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV:Surface Probability Plot—Emini.

DETAILED DESCRIPTION

[0033] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a follistatin-3 polypeptide havingthe amino acid sequence shown in SEQ ID NO:2, which was determined bysequencing a cloned cDNA. The nucleotide sequence shown in FIGS. 1A, 1B,and 1C (SEQ ID NO:1) was obtained by sequencing the HDTAH85 clone, whichwas deposited on Aug. 8, 1997 at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, and givenaccession number ATCC® 209199. The deposited clone is contained in thepBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.).

[0034] The follistatin-3 protein of the present invention sharessequence homology with the translation product of the human mRNA forfollistatin-1 (FIG. 2; SEQ ID NO:3). Follistatin-1 is thought to be animportant factor in the regulation of follicle development andspermatogenesis in the reproductive systems. Follistatin-1 acts as anantagonist of activin by stoichiometrically binding to activin andpreventing interaction with the activin receptor. It is thought that, inaddition to activin, follistatin-1 may act in a similar manner bytargeting additional members of the TGF-beta superfamily. Nucleic AcidMolecules

[0035] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.,Foster City, Calif.), and all amino acid sequences of polypeptidesencoded by DNA molecules determined herein were predicted by translationof a DNA sequence determined as above. Therefore, as is known in the artfor any DNA sequence determined by this automated approach, anynucleotide sequence determined herein may contain some errors.Nucleotide sequences determined by automation are typically at leastabout 90% identical, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of the sequenced DNAmolecule. The actual sequence can be more precisely determined by otherapproaches including manual DNA sequencing methods well known in theart. As is also known in the art, a single insertion or deletion in adetermined nucleotide sequence compared to the actual sequence willcause a frame shift in translation of the nucleotide sequence such thatthe predicted amino acid sequence encoded by a determined nucleotidesequence will be completely different from the amino acid sequenceactually encoded by the sequenced DNA molecule, beginning at the pointof such an insertion or deletion.

[0036] By “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U), where each thyrnidine deoxyribonucleotide (T) in the specifieddeoxyribonucleotide sequence is replaced by the ribonucleotide uridine(U).

[0037] Using the information provided herein, such as the nucleotidesequence in FIGS. 1A, 1B, and 1C (SEQ ID NO:1), a nucleic acid moleculeof the present invention encoding a follistatin-3 polypeptide may beobtained using standard cloning and screening procedures, such as thosefor cloning cDNAs using mRNA as starting material. Illustrative of theinvention, the nucleic acid molecule described in FIGS. 1A, 1B, and 1C(SEQ ID NO:1) was discovered in a cDNA library derived from Hodgkin'sLymphoma.

[0038] Additional clones of the same gene were also identified in cDNAlibraries from the following cells and tissues: synovial fibroblasts,gall bladder, resting and serum-induced smooth muscle, testes, Merkelcells, HEL cells, hippocampus, TNF-alpha- and IFN-induced epithelialcells, keratinocyte, amygdala depression, HL-60 cells, hepatoma,progesterone-treated epidermal cells, endothelial cells, HSC172 cells,epithelioid sarcoma, activated T-cells, breast lymph node, pancreaticcarcinoma, fetal dura mater, fetal lung, epididymis, placenta, dendriticcells, rejected kidney, and uterine cancer.

[0039] The determined nucleotide sequence of the follistatin-3 cDNA ofFIGS. 1A, 1B, and 1C (SEQ ID NO:1) contains an open reading frameencoding a protein of 263 amino acid residues, with an initiation codonat nucleotide positions 19-21 of the nucleotide sequence in FIG. 1A (SEQID NO:1), and a deduced molecular weight of about 27.7 kDa. The aminoacid sequence of the follistatin-3 protein shown in SEQ ID NO:2 is about43.2% identical to human mRNA for follistatin-1 (FIG. 2; Shimasaki, S.,et al., Proc. Natl. Acad. Sci. U.S.A. 85:4218-4222 (1988); GenBankAccession No. J03771).

[0040] The open reading frame of the follistatin-3 gene shares sequencehomology with the translation product of the human mRNA forfollistatin-1 (FIG. 2; SEQ ID NO:3). The homology between follistatin-1and follistatin-3 indicates that follistatin-3 may also be involved in aphysiological regulation of cell growth and differentiation,particularly with regard to cells of the reproductive system.

[0041] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors discussed above, the actual completefollistatin-3 polypeptide encoded by the deposited cDNA, which comprisesabout 263 amino acids, may be somewhat longer or shorter. Moregenerally, the actual open reading frame may be anywhere in the range of±20 amino acids, more likely in the range of ±10 amino acids, of thatpredicted from either the methionine codon from the N-terminus shown inFIG. 1A (SEQ ID NO:1). It will further be appreciated that, depending onthe analytical criteria used for identifying various functional domains,the exact “address” of the mature form of the follistatin-3 polypeptidemay differ slightly from the predicted positions above. For example, theexact location of the cleavage site of the precursor form of the maturefollistatin-3 molecule shown in SEQ ID NO:2 may vary slightly (e.g., theaddress may “shift” by about 6 residues, depending on the criteria usedto define the cleavage site. In this case, the ends of the signalpeptide and the beginning of the mature follistatin-3 molecule werepredicted using the HGSI SignalP computer algorithm. One of skill in theart will realize that another widely accepted computer algorithm used topredict potential sites of polypeptide cleavage, PSORT, will predict thecleavage of an N-terminal signal peptide from the follistatin-3polypeptide at a point slightly different from that predicted by theHGSI SignalP algorithm. In either case, as discussed further below, theinvention further provides polypeptides having various residues deletedfrom the N-terminus of the complete polypeptide, including polypeptidescorresponding to either of the predicted mature follistatin-3polypeptides described herein.

[0042] The amino acid sequence of the complete follistatin-3 proteinincludes a leader sequence and a mature protein, as shown in SEQ IDNO:2. More in particular, the present invention provides nucleic acidmolecules encoding a mature form of the follistatin-3 protein. Thus,according to the signal hypothesis, once export of the growing proteinchain across the rough endoplasmic reticulum has been initiated,proteins secreted by mammalian cells have a signal or secretory leadersequence which is cleaved from the complete polypeptide to produce asecreted “mature” form of the protein. Most mammalian cells and eveninsect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species of the protein.Further, it has long been known that the cleavage specificity of asecreted protein is ultimately determined by the primary structure ofthe complete protein, that is, it is inherent in the amino acid sequenceof the polypeptide. Therefore, the present invention provides anucleotide sequence encoding the mature follistatin-3 polypeptide havingthe amino acid sequence encoded by the cDNA clone contained in the hostidentified as ATCC® Deposit No. 209199. By the “mature follistatin-3polypeptide having the amino acid sequence encoded by the cDNA clone inATCC® Deposit No. 209199” is meant the mature form(s) of thefollistatin-3 protein produced by expression in a mammalian cell (e.g.,COS cells, as described below) of the complete open reading frameencoded by the human DNA sequence of the clone contained in the vectorin the deposited host.

[0043] In addition, methods for predicting whether a protein has asecretory leader as well as the cleavage point for that leader sequenceare available. For instance, the method of McGeoch (Virus Res. 3:271-286(1985)) uses the information from a short N-terminal charged region anda subsequent uncharged region of the complete (uncleaved) protein. Themethod of von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) uses theinformation from the residues surrounding the cleavage site, typicallyresidues −13 to +2 where +1 indicates the amino terminus of the matureprotein. The accuracy of predicting the cleavage points of knownmammalian secretory proteins for each of these methods is in the rangeof 75-80% (von Heinje, supra). However, the two methods do not alwaysproduce the same predicted cleavage point(s) for a given protein.

[0044] In the present case, the deduced amino acid sequence of thecomplete follistatin-3 polypeptide was analyzed by a variation of thecomputer program “PSORT”, available from Dr. Kenta Nakai of theInstitute for Chemical Research, Kyoto University (Nakai, K. andKanehisa, M. Genomics 14:897-911 (1992)), which is an expert system forpredicting the cellular location of a protein based on the amino acidsequence. As part of this computational prediction of localization, themethods of McGeoch and von Heinje are incorporated. Thus, thecomputation analysis above predicted a single cleavage site within thecomplete amino acid sequence shown in SEQ ID NO:2 (see abovediscussion).

[0045] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0046] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

[0047] Isolated nucleic acid molecules of the present invention includeDNA molecules comprising an open reading frame (ORF) with an initiationcodon at positions 19-21 of the nucleotide sequence shown in FIG. 1A(SEQ ID NO:1).

[0048] Also included are DNA molecules comprising the coding sequencefor the predicted mature follistatin-3 protein shown at positions 1-237of SEQ ID NO:2.

[0049] In addition, isolated nucleic acid molecules of the inventioninclude DNA molecules which comprise a sequence substantially differentfrom those described above but which, due to the degeneracy of thegenetic code, still encode the follistatin-3 protein. Of course, thegenetic code and species-specific codon preferences are well known inthe art. Thus, it would be routine for one skilled in the art togenerate the degenerate variants described above, for instance, tooptimize codon expression for a particular host (e.g., change codons inthe human mRNA to those preferred by a bacterial host such as E. coli).

[0050] In another aspect, the invention provides isolated nucleic acidmolecules encoding the follistatin-3 polypeptide having an amino acidsequence encoded by the cDNA clone contained in the plasmid deposited asATCC® Deposit No. 209199 on Aug. 8, 1997.

[0051] Preferably, this nucleic acid molecule will encode the maturepolypeptide encoded by the above-described deposited cDNA clone.

[0052] The invention further provides an isolated nucleic acid moleculehaving the nucleotide sequence shown in FIGS. 1A, 1B, and 1C (SEQ IDNO:1) or the nucleotide sequence of the follistatin-3 cDNA contained inthe above-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences. Such isolatedmolecules, particularly DNA molecules, are useful as probes for genemapping, by in situ hybridization with chromosomes, and for detectingexpression of the follistatin-3 gene in human tissue, for instance, byNorthern blot analysis.

[0053] The present invention is further directed to nucleic acidmolecules encoding portions of the nucleotide sequences described hereinas well as to fragments of the isolated nucleic acid molecules describedherein. In particular, the invention provides a polynucleotide having anucleotide sequence representing the portion of SEQ ID NO:1 whichconsists of positions 1-810 of SEQ ID NO:1.

[0054] In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 whichhave been determined from the following related cDNA clones: HHPDX66R(SEQ ID NO:4), HDTAH61R (SEQ ID NO:5), HSBAV55R (SEQ ID NO:6), HUKFS32R(SEQ ID NO:7), HOOAD78R (SEQ ID NO:8), HAQAG52R (SEQ ID NO:9), HTLEJ56R(SEQ ID NO:10), HLMNX90R (SEQ ID NO:11).

[0055] Further, the invention includes a polynucleotide comprising anyportion of at least about 30 nucleotides, preferably at least about 50nucleotides, of SEQ ID NO:1 from residue 1 to 500. More preferably, theinvention includes a polynucleotide comprising nucleotide residues100-500, 200-500, 300-500, 400-500, 100-400, 200-400, 300-400, 100-300,200-300, 100-200, 100-2495, 250-2495, 500-2495, 1000-2495, 1500-2495,2000-2495, 100-2000, 250-2000, 500-2000, 1000-2000, 1500-2000, 100-1500,250-1500, 500-1500, 1000-1500, 100-1000, 250-1000, and 500-1000.

[0056] More generally, by a fragment of an isolated nucleic acidmolecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) isintended fragments at least about 15 nt, and more preferably at leastabout 20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50-300 nt in length are also useful according to the presentinvention as are fragments corresponding to most, if not all, of thenucleotide sequence of the deposited cDNA or as shown in FIGS. 1A, 1B,and 1C (SEQ ID NO:1). By a fragment at least 20 nt in length, forexample, is intended fragments which include 20 or more contiguous basesfrom the nucleotide sequence of the deposited cDNA or the nucleotidesequence as shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1). Preferrednucleic acid fragments of the present invention include nucleic acidmolecules encoding epitope-bearing portions of the follistatin-3polypeptide as identified in FIG. 3 and described in more detail below.

[0057] In specific embodiments, the polynucleotide fragments of theinvention encode a polypeptide which demonstrates a functional activity.By a polypeptide demonstrating “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a complete, mature or active form of thefollistatin-3 polypeptide. Such functional activities include, but arenot limited to, biological activity ((e.g., modulating the folliclestimulating hormone (FSH) synthetic pathway, increasing estradiolproduction, binding activin, stimulator of gonadotropin biosynthesis andsecretion, regulator of ovarian and placental steroidogenesis, andoocyte and spermatogonial maturation factor)), antigenicity [ability tobind (or compete with a follistatin-3 polypeptide for binding) to ananti-follistatin-3 antibody], immunogenicity (ability to generateantibody which binds to a follistatin-3 polypeptide), the ability toform polymers with other follistatin-3 or inhibin or TGF-betapolypeptides, and ability to bind to a receptor or ligand for afollistatin-3 polypeptide.

[0058] Preferred nucleic acid fragments of the present invention alsoinclude nucleic acid molecules encoding one or more of the followingdomains of follistatin-3: amino acid residues 7-16, 34-45, 78-86,91-100, 108-122, 131-145, 156-169, 184-192, and 196-210 of SEQ ID NO:2.

[0059] In specific embodiments, the polynucleotide fragments of theinvention encode antigenic regions. Non-limiting examples of antigenicpolypeptides or peptides that can be used to generatefollistatin-3-specific antibodies include: a polypeptide comprisingamino acid residues from about Leu-14 to about Ala-20, from about Ser-46to about Ile-55, from about Gly-88 to about Pro-97, from about Gly-113to about Leu-133, from about Arg-138 to about Glu-146, from aboutPro-177 to about Thr-191, from about Gly-219 to about Val-237.

[0060] In additional embodiments, the polynucleotides of the inventionencode functional attributes of follistatin-3. Preferred embodiments ofthe invention in this regard include fragments that comprise alpha-helixand alpha-helix forming regions (“alpha-regions”), beta-sheet andbeta-sheet forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions and high antigenic index regions offollistatin-3.

[0061] The data representing the structural or functional attributes offollistatin-3 set forth in FIG. 3 and/or Table I, as described above,was generated using the various modules and algorithms of the DNA*STARset on default parameters. In a preferred embodiment, the data presentedin columns VIII, IX, XIII, and XIV of Table I can be used to determineregions of follistatin-3 which exhibit a high degree of potential forantigenicity. Regions of high antigenicity are determined from the datapresented in columns VIII, IX, XIII, and/or IV by choosing values whichrepresent regions of the polypeptide which are likely to be exposed onthe surface of the polypeptide in an environment in which antigenrecognition may occur in the process of initiation of an immuneresponse.

[0062] Certain preferred regions in these regards are set out in FIG. 3,but may, as shown in Table I, be represented or identified by usingtabular representations of the data presented in FIG. 3. The DNA*STARcomputer algorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table I). The tabular format of the data in FIG. 3 may be used toeasily determine specific boundaries of a preferred region.

[0063] The above-mentioned preferred regions set out in FIG. 3 and inTable I include, but are not limited to, regions of the aforementionedtypes identified by analysis of the amino acid sequence set out in FIGS.1A, 1B, and 1C. As set out in FIG. 3 and in Table I, such preferredregions include Garnier-Robson alpha-regions, beta-regions,turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions,and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobicregions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulzflexible regions, Emini surface-forming regions and Jameson-Wolf regionsof high antigenic index. TABLE I Res Position I II III IV V VI VII VIIIIX X XI XII XIII XIV Met 1 . . B . . . . 0.31 −0.24 * * . 1.07 1.11 Arg2 . . B . . . . 0.49 −0.17 * * . 1.13 0.88 Pro 3 . . . . T . . 0.53−0.17 * * . 1.89 1.06 Gly 4 . . . . T . . 0.71 −0.17 * * . 2.10 1.06 Ala5 . . . . . T C 0.29 −0.36 . * F 1.89 0.84 Pro 6 . . . . . T C 0.60 0.33. * F 1.08 0.45 Gly 7 . . . . . T C 0.28 0.81 . * F 0.57 0.48 Pro 8 . .B . . T . −0.32 0.81 . . F 0.16 0.73 Leu 9 . . B . . . . −0.19 1.00 . .F −0.25 0.39 Trp 10 . . B . . . . 0.11 1.00 . . . −0.40 0.61 Pro 11 . .B . . . . −0.02 1.49 . . . −0.40 0.41 Leu 12 . . B . . T . −0.27 1.49 .. . −0.20 0.49 Pro 13 . . . . T T . −0.87 1.30 . . . 0.20 0.48 Trp 14 .. . . T T . −0.64 1.07 . . . 0.20 0.25 Gly 15 . . . . . T C −0.64 1.14 .. . 0.00 0.31 Ala 16 . A . . . . C −1.02 1.37 . . . −0.40 0.21 Leu 17 .A B . . . . −1.07 1.44 . . . −0.60 0.20 Ala 18 . A B B . . . −1.20 1.17. . . −0.60 0.15 Trp 19 . A B B . . . −1.61 1.17 . . . −0.60 0.15 Ala 20. A B B . . . −2.12 1.46 . . . −0.60 0.16 Val 21 . A B B . . . −1.831.41 . . . −0.60 0.11 Gly 22 . A B B . . . −1.32 1.30 . . . −0.60 0.15Phe 23 . . B B . . . −1.33 0.77 . . . −0.60 0.19 Val 24 . . B B . . .−1.39 0.89 . . . −0.60 0.26 Ser 25 . . B . . . . −1.10 0.67 * . . −0.400.26 Ser 26 . . B . . . . −0.59 0.63 . . F −0.25 0.40 Met 27 . . . . T .. −0.24 0.27 . . F 0.45 0.53 Gly 28 . . . . T T . 0.24 0.03 . . F 0.820.64 Ser 29 . . . . T T . 0.51 0.07 . . F 0.99 0.74 Gly 30 . . . . . T C0.60 0.19 . . F 0.96 0.76 Asn 31 . . . . . T C 0.56 −0.00 . . F 1.881.18 Pro 32 . . . . . . C 0.81 −0.00 . . F 1.70 0.87 Ala 33 . . . . . TC 0.30 0.04 . . F 1.13 0.87 Pro 34 . . . . T T . −0.07 0.26 . . F 1.160.40 Gly 35 . . . . T T . −0.01 0.43 * . F 0.69 0.14 Gly 36 . . B . . T. −0.82 0.91 * . F 0.12 0.15 Val 37 . A B . . . . −0.61 1.10 * . . −0.600.08 Cys 38 . A B . . . . −0.02 1.07 * . . −0.60 0.14 Trp 39 . A B . . .. −0.16 1.04 * . . −0.60 0.24 Leu 40 . A B . . . . 0.19 1.04 * . . −0.320.32 Gln 41 . . B . . T . 0.53 0.80 * . F 0.66 1.02 Gln 42 . . . . T T .0.80 0.23 * . F 1.64 1.68 Gly 43 . . . . T T . 1.16 −0.19 * . F 2.522.06 Gln 44 . . . . T T . 0.78 −0.39 * . F 2.80 1.72 Glu 45 . . . . T .. 1.29 −0.21 * . F 2.17 0.53 Ala 46 . . . . T T . 0.48 −0.23 * . F 2.090.72 Thr 47 . . B . . T . −0.38 0.03 . . . 0.66 0.34 Cys 48 . . B . . T. −0.84 0.27 . . . 0.38 0.15 Ser 49 . . B . . T . −0.84 0.96 . . . −0.200.12 Leu 50 . . B B . . . −1.16 0.86 . * . −0.60 0.14 Val 51 . . B B . .. −0.57 0.86 . * . −0.60 0.39 Leu 52 . . B B . . . −1.11 0.29 . * .−0.30 0.48 Gln 53 . . B B . . . −0.76 0.54 * * F −0.45 0.43 Thr 54 . . BB . . . −0.34 0.34 * . F −0.15 0.85 Asp 55 . . B B . . . −0.12 −0.30 * *F 0.60 2.01 Val 56 . A B B . . . 0.73 −0.49 * . F 0.60 1.17 Thr 57 . A BB . . . 0.88 0.89 * * F 0.90 1.41 Arg 58 . A B B . . . 0.21 −0.80 * * F0.75 0.45 Ala 59 . A B B . . . −0.07 −0.23 * * . 0.30 0.33 Glu 60 . A BB . . . −0.37 −0.37 * * . 0.30 0.23 Cys 61 . A B . . . . 0.14 −0.47 * *. 0.55 0.16 Cys 62 . . . . T T . 0.46 −0.04 * * . 1.60 0.15 Ala 63 . . .. T T . −0.54 −0.14 * * . 1.85 0.14 Ser 64 . . . . T T . 0.04 0.54 . * F1.35 0.19 Gly 65 . . . . T T . −0.27 −0.03 . * F 2.50 0.58 Asn 66 . . .. T T . −0.19 −0.11 . * F 2.25 0.83 Ile 67 . . B . . T . 0.19 −0.11 * *F 1.60 0.62 Asp 68 . . B . . T . 0.48 0.41 * * F 0.45 0.66 Thr 69 . . B. . T . 0.78 0.37 * * F 0.50 0.55 Ala 70 . . B . . . . 0.31 0.37 * * .0.05 1.26 Trp 71 . . B . . T . −0.00 0.37 * . . 0.10 0.62 Ser 72 . . B .. T . 0.86 0.86 * . . −0.20 0.62 Asn 73 . . B . . T . 0.64 0.87 * . .−0.20 0.84 Leu 74 . . . . . T C 0.61 0.80 * . . 0.43 1.24 Thr 75 . . . .. . C 1.20 0.31 * . . 0.66 0.91 His 76 . . . . . T C 1.53 0.33 * . F1.29 0.91 Pro 77 . . . . . T C 0.94 −0.07 * . F 2.32 2.22 Gly 78 . . . .T T . 0.94 −0.07 * * F 2.80 1.08 Asn 79 . . . . T T . 0.94 −0.16 * . F2.52 1.27 Lys 80 . . B . . . . 0.44 0.03 * * F 0.89 0.68 Ile 81 . . B .. . . 0.13 0.29 . . F 0.61 0.57 Asn 82 . . B . . . . −0.36 0.29 . * .0.18 0.35 Leu 83 . . B B . . . −0.82 0.67 . . . −0.60 0.15 Leu 84 . . BB . . . −1.17 1.36 . * . −0.60 0.18 Gly 85 . . B B . . . −2.02 1.10 . *. −0.60 0.11 Phe 86 . . B B . . . −1.99 1.39 . . . −0.60 0.11 Leu 87 . .B B . . . −2.02 1.34 . . . −0.60 0.10 Gly 88 . . B B . . . −1.88 1.16 *. . −0.60 0.13 Leu 89 . . B B . . . −1.88 1.30 . . . −0.60 0.08 Val 90 .. B B . . . −1.74 1.20 * . . −0.60 0.08 His 91 . . B B . . . −1.710.94 * . . −0.60 0.13 Cys 92 . . B B . . . −0.86 1.09 . . . −0.60 0.08Leu 93 . . B B . . . −0.51 0.40 . . . 0.01 0.23 Pro 94 . . . B T . .−0.00 −0.24 . . . 1.32 0.28 Cys 95 . . . . T T . 0.19 −0.36 . . . 2.030.70 Lys 96 . . . . T T . 0.22 −0.36 . . F 2.49 0.46 Asp 97 . . . . T T. 0.54 −1.04 * . F 3.10 0.49 Ser 98 . . . . T T . 0.50 −1.04 * . F 2.790.91 Cys 99 . . . . T T . 0.71 −0.97 * . F 2.48 0.34 Asp 100 . . B . . T. 0.71 −0.97 * . F 1.77 0.35 Gly 101 . . B . . T . 0.32 −0.40 * . F 1.470.14 Val 102 . . B . . T . 0.11 −0.36 * . . 1.32 0.26 Glu 103 . . B . .. . 0.07 −0.50 * . . 1.73 0.24 Cys 104 . . . . T . . 0.78 −0.07 * . F2.29 0.24 Gly 105 . . . . T T . 0.19 −0.50 * . F 3.10 0.64 Pro 106 . . .. T T . −0.13 −0.64 * . F 2.79 0.38 Gly 107 . . . . T T . 0.83 −0.07 * .F 2.18 0.38 Lys 108 . . . . T T . 0.23 −0.64 * . F 2.17 0.74 Ala 109 . AB . . . . 0.09 −0.46 * . . 0.61 0.48 Cys 110 . A B . . . . 0.09 −0.20 *. . 0.30 0.40 Arg 111 . A B . . . . −0.04 −0.20 * . . 0.30 0.20 Met 112. A B . . . . 0.41 0.23 * . . −0.30 0.19 Leu 113 . A . . T . . 0.16−0.27 * * . 1.04 0.70 Gly 114 . A . . T . . 0.86 −0.41 * * F 1.53 0.55Gly 115 . . . . T . . 0.86 −0.41 * * F 2.22 1.10 Arg 116 . . . . . T C0.74 −0.46 * * F 2.41 0.71 Pro 117 . . . . T T . 0.68 −1.14 . * F 3.401.25 Arg 118 . . . . T T . 0.90 −1.00 . * F 2.91 0.68 Cys 119 . . B . .T . 1.03 −0.93 . * . 2.02 0.35 Glu 120 . . B . . . . 1.38 −0.50 . * .1.73 0.35 Cys 121 . . B . . . . 0.60 −0.93 . * . 1.64 0.30 Ala 122 . . B. . T . 0.51 −0.36 . * . 1.45 0.30 Pro 123 . . . . T T . 0.06 −0.54 . *F 2.55 0.23 Asp 124 . . . . T T . −0.09 −0.11 . . F 2.50 0.43 Cys 125 .. . . T T . −0.30 −0.00 . . F 2.25 0.35 Ser 126 . . . . T . . −0.22−0.07 * * F 1.80 0.35 Gly 127 . . . . T . . 0.48 −0.00 * * F 1.55 0.21Leu 128 . . B . . . . −0.12 −0.00 * * . 0.75 0.77 Pro 129 . . B . . . .−0.12 0.11 . * . −0.10 0.47 Ala 130 . . B . . . . −0.31 0.13 . * . −0.100.83 Arg 131 . . B B . . . −0.68 0.34 . * . −0.30 0.74 Leu 132 . . B B .. . −0.68 0.23 . * . −0.30 0.26 Gln 133 . . B B . . . −0.17 0.23 * * .−0.30 0.25 Val 134 . . B B . . . 0.04 0.11 * * . −0.30 0.17 Cys 135 . .B B . . . 0.29 0.11 * * . −0.02 0.35 Gly 136 . . B . . T . −0.41−0.14 * * F 1.41 0.20 Ser 137 . . . . T T . 0.09 −0.04 . . F 2.09 0.27Asp 138 . . . . T T . −0.16 −0.20 . * F 2.37 0.73 Gly 139 . . . . T T .0.81 −0.01 . . F 2.80 1.16 Ala 140 . . . . T . . 1.48 −0.44 . . F 2.321.70 Thr 141 . . B . . . . 1.82 −0.83 . . . 1.99 1.70 Tyr 142 . . B . .T . 1.46 −0.83 . * . 2.11 2.97 Arg 143 . . B . . T . 1.46 −0.69 . * F2.18 1.57 Asp 144 . . B . . T . 0.99 −1.19 . * F 2.10 1.89 Glu 145 . . B. . T . 1.69 −0.99 . * . 2.00 0.99 Cys 146 . A B . . . . 1.41 −1.74 . *. 1.40 0.99 Glu 147 A A . . . . . 1.07 −1.24 . * . 1.20 0.60 Leu 148 A A. . . . . 1.07 −0.74 . * . 1.00 0.35 Arg 149 A A . . . . . 0.40 −0.74. * . 0.95 1.28 Ala 150 A A . . . . . 0.51 −0.74 * * . 0.60 0.40 Ala 151. A . . T . . 0.83 −0.74 . * . 1.00 0.94 Arg 152 . A . . T . . 0.80−1.00 . * . 1.00 0.48 Cys 153 . A . . T . . 1.40 −0.50 . * . 1.27 0.64Arg 154 . A . . T . . 1.29 −0.57 . * . 1.54 0.98 Gly 155 . A . . T . .1.07 −1.07 . * F 1.96 0.84 His 156 . . . . . T C 1.36 −0.39 . * F 2.281.29 Pro 157 . . . . . T C 0.39 −0.57 . * F 2.70 0.88 Asp 158 . . . . TT . 0.46 0.07 * * F 1.73 0.66 Leu 159 . . B . . T . 0.10 0.26 * * . 0.910.48 Ser 160 . . B B . . . 0.56 0.51 * * . −0.06 0.49 Val 161 . . B B .. . 0.24 0.09 . * . −0.03 0.57 Met 162 . . B B . . . 0.57 0.51 . * .−0.26 0.69 Tyr 163 . . B . . T . −0.10 −0.17 * * . 1.53 1.00 Arg 164 . .B . . T . 0.82 0.01 . * . 1.12 0.72 Gly 165 . . . . T T . 1.17 −0.63 . *F 3.06 1.43 Arg 166 . . . . T T . 1.72 −1.24 . * F 3.40 1.83 Cys 167 . .. . T . . 1.66 −1.61 * * F 2.86 1.25 Arg 168 . . . . T T . 1.90−1.04 * * F 2.57 0.68 Lys 169 . . . . T T . 1.76 −1.47 * * F 2.23 0.60Ser 170 . . . . T T . 1.24 −0.97 * * F 2.04 1.52 Cys 171 . . . . T T .0.28 −0.90 * * . 1.40 0.58 Glu 172 . . B B . . . 0.28 −0.26 * . . 0.300.21 His 173 . . B B . . . −0.04 0.31 . . . −0.30 0.09 Val 174 . . B B .. . 0.02 0.36 . * . −0.02 0.25 Val 175 . . B B . . . 0.11 −0.21 . * .0.86 0.28 Cys 176 . . B . . T . 0.78 0.21 . * . 0.94 0.32 Pro 177 . . .. T T . 0.48 0.11 . . F 1.77 0.74 Arg 178 . . . . T T . −0.16 −0.14 . *F 2.80 1.34 Pro 179 . . . . T T . −0.16 −0.21 . * F 2.52 1.34 Gln 180 .. . B T . . −0.16 −0.14 * * F 1.69 0.64 Ser 181 . . B B . . . 0.510.07 * * F 0.41 0.24 Cys 182 . . B B . . . 0.72 0.07 * * . −0.02 0.26Val 183 . . B B . . . 0.30 0.04 * * . −0.30 0.26 Val 184 . . B B . . .0.17 0.13 . . . −0.02 0.28 Asp 185 . . B B . . . −0.13 0.17 . . F 0.410.52 Gln 186 . . B . . T . −0.42 −0.01 . . F 1.69 0.94 Thr 187 . . . . TT . 0.21 −0.16 . . F 2.52 1.28 Gly 188 . . . . T T . 0.40 −0.30 . . F2.80 1.04 Ser 189 . . . . T T . 0.40 0.27 . . F 1.77 0.32 Ala 190 . . BB T . . −0.46 0.51 . . . 0.64 0.17 His 191 . . B B . . . −1.12 0.67 * .. −0.04 0.12 Cys 192 . . B B . . . −0.70 0.81 * * . −0.32 0.05 Val 193 .. B B . . . −0.94 0.43 * . . −0.60 0.10 Val 194 . . B B . . . −1.230.43 * . . −0.60 0.07 Cys 195 . . B B . . . −0.86 0.43 * . . −0.60 0.14Arg 196 . . B B . . . −1.49 0.29 * . . −0.30 0.28 Ala 197 . . B B . . .−1.03 0.21 * . . −0.30 0.20 Ala 198 . . B . . T . −1.03 −0.00 * . . 0.700.59 Pro 199 . . B . . T . −0.39 0.07 * . . 0.10 0.22 Cys 200 . . B . .T . −0.02 0.50 * . . −0.20 0.34 Pro 201 . . B . . T . −0.43 0.39 * . .0.10 0.45 Val 202 . . B . . . . −0.06 0.27 . . F 0.05 0.39 Pro 203 . . .. T . . 0.19 0.27 . . F 0.88 1.13 Ser 204 . . . . T . . 0.40 0.13 . . F1.01 0.72 Ser 205 . . . . . T C 1.07 0.10 * . F 1.44 1.69 Pro 206 . . .. T T . 0.47 −0.54 * . F 2.82 1.89 Gly 207 . . . . T T . 0.66 −0.29 . .F 2.80 1.16 Gln 208 . . B . . T . 0.52 −0.10 . . F 1.97 0.47 Glu 209 . .B . . . . 0.82 −0.06 . . F 1.49 0.30 Leu 210 . . B . . . . 1.12 −0.09 .. F 1.37 0.48 Cys 211 . . B . . T . 1.33 −0.11 . . F 1.45 0.45 Gly 212 .. . . T T . 0.82 −0.11 . . F 1.73 0.42 Asn 213 . . . . T T . 0.51 0.53. * F 0.99 0.38 Asn 214 . . . . T T . 0.27 0.33 . * F 1.60 1.01 Asn 215. . . B T . . 0.19 0.51 . . F 0.74 1.60 Val 216 . . B B . . . 0.560.77 * . . −0.12 0.70 Thr 217 . . B B . . . 0.60 0.76 * . . −0.28 0.58Tyr 218 . . B B . . . −0.07 0.74 . . . −0.44 0.48 Ile 219 . . B B . . .−0.10 0.91 . . . −0.60 0.35 Ser 220 . . B . . T . −0.70 0.77 * * . −0.200.33 Ser 221 . . B . . T . 0.27 0.90 * . . −0.20 0.21 Cys 222 . . B . .T . 0.58 0.14 * . . 0.10 0.58 His 223 . . B . . T . 0.23 −0.14 * . .0.70 0.75 Met 224 . . . . T . . 0.81 −0.03 * . . 0.90 0.57 Arg 225 . . BB . . . 0.44 0.07 * * . −0.15 1.53 Gln 226 . . B B . . . 0.04 0.07 * . .−0.30 0.60 Ala 227 . . B B . . . −0.10 0.36 * . . −0.30 0.53 Thr 228 . .B B . . . −0.41 0.43 * * . −0.60 0.22 Cys 229 . . B B . . . 0.300.86 * * . −0.60 0.13 Phe 230 . . B B . . . −0.11 046 * . . −0.60 0.25Leu 231 . . B B . . . −1.00 0.34 * . . −0.30 0.23 Gly 232 . . . . T T .−0.76 0.54 * . . 0.20 0.30 Arg 233 . . . . T T . −1.30 0.40 . * F 0.650.34 Ser 234 . . . . T T . −0.52 0.26 . . F 0.65 0.31 Ile 235 . . B . .T . 0.14 −0.43 . . . 0.70 0.61 Gly 236 . . B B . . . 0.37 −0.36 . . .0.30 0.42 Val 237 . . B B . . . 0.37 0.14 . . . −0.30 0.32 Arg 238 . . BB . . . −0.04 0.19 . * . −0.30 0.45 His 239 . . B . . T . −0.41−0.11 * * . 0.70 0.61 Ala 240 . . . . T T . −0.11 0.03 * * . 0.50 0.44Gly 241 . . . . T T . −0.11 −0.11 * . . 1.10 0.23 Ser 242 . . . . T T .0.43 0.31 * * . 0.80 0.16 Cys 243 . . . . T T . 0.11 0.30 * . . 1.100.24 Ala 244 . . . . T T . 0.14 0.23 . . . 1.40 0.37 Gly 245 . . . . . TC 0.73 −0.20 . . F 2.25 0.47 Thr 246 . . . . . T C 0.87 −0.59 . . F 3.001.53 Pro 247 . . . . . . C 0.96 −0.73 . . F 2.50 2.35 Glu 248 . . . . .. C 1.28 −0.80 . . F 2.50 3.67 Glu 249 . . . . . . C 1.52 −0.80 . . F2.50 2.52 Pro 250 . . . . . T C 1.87 −0.86 . . F 2.70 1.61 Pro 251 . . .. . T C 1.88 −1.29 . . F 2.70 1.61 Gly 252 . . . . . T C 1.50 −0.90 . .F 3.00 1.25 Gly 253 . . . . . T C 1.50 −0.40 . . F 2.25 0.81 Glu 254 . A. . . . C 1.50 −0.83 . . F 1.85 0.91 Ser 255 . A . . . . C 1.71 −1.26 .. F 1.70 1.60 Ala 256 A A . . . . . 1.92 −1.69 * . F 1.20 2.79 Glu 257 AA . . . . . 2.27 −2.11 * . F 0.90 2.79 Glu 258 A A . . . . . 1.91−1.71 * . F 0.90 3.35 Glu 259 A A . . . . . 1.06 −1.31 * . F 0.90 2.87Glu 260 A A . . . . . 0.97 −1.17 * . F 0.90 1.23 Asn 261 A A . . . . .1.17 −0.74 * . . 0.60 0.91 Phe 262 A A . . . . . 0.78 −0.31 . . . 0.300.67 Val 263 A A . . . . . 0.39 0.11 . . . −0.30 0.49

[0064] Among highly preferred fragments in this regard are those thatcomprise reigons of follistatin-3 that combine several structuralfeatures, such as several of the features set out above.

[0065] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a portion of the polynucleotide ina nucleic acid molecule of the invention described above, for instance,the cDNA clone contained in ATCC® Deposit No. 209199. By “stringenthybridization conditions” is intended overnight incubation at 42 C in asolution comprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 micrograms/ml denatured, sheared salmon spermDNA, followed by washing the filters in 0.1×SSC at about 65 C.

[0066] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 (e.g., 50) nt of the referencepolynucleotide. These are useful as diagnostic probes and primers asdiscussed above and in more detail below.

[0067] By a portion of a polynucleotide of “at least 20 nt in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIGS. 1A, 1B, and 1C (SEQ IDNO:1)). Of course, a polynucleotide which hybridizes only to a poly Asequence (such as the 3′ terminal poly(A) tract of the follistatin-3cDNA shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1)), or to a complementarystretch of T (or U) residues, would not be included in a polynucleotideof the invention used to hybridize to a portion of a nucleic acid of theinvention, since such a polynucleotide would hybridize to any nucleicacid molecule containing a poly (A) stretch or the complement thereof(e.g., practically any double-stranded cDNA clone).

[0068] In preferred embodiments, polynucleotides which hybridize to thereference polynucleotides disclosed herein encode polypeptides whicheither retain substantially the same biological function or activity asthe mature form of the follistatin-3 polypeptide encoded by thepolynucleotide sequence depicted in FIGS. 1A, 1B, and 1C (SEQ ID NO:1)or the clone contained in the deposit (HDTAH85).

[0069] Alternative embodiments are directed to polynucleotides whichhybridize to the reference polynucleotide (i.e., a polynucleotidesequence disclosed herein), but do not retain biological activity. Whilethese polynucleotides do not retain biological activity, they have uses,such as, for example, as probes for the polynucleotides of SEQ ID NO:1,for recovery of the polynucleotides, as diagnostic probes, and as PCRprimers.

[0070] As indicated, nucleic acid molecules of the present inventionwhich encode a follistatin-3 polypeptide may include, but are notlimited to those encoding the amino acid sequence of the maturepolypeptide, by itself; and the coding sequence for the maturepolypeptide and additional sequences, such as those encoding the about26 amino acid leader or secretory sequence, such as a pre-, or pro- orprepro-protein sequence; the coding sequence of the mature polypeptide,with or without the aforementioned additional coding sequences.

[0071] Also encoded by nucleic acids of the invention are the aboveprotein sequences together with additional, non-coding sequences,including for example, but not limited to introns and non-coding 5′ and3′ sequences, such as the transcribed, non-translated sequences thatplay a role in transcription, mRNA processing, including splicing andpolyadenylation signals, for example—ribosome binding and stability ofmRNA; an additional coding sequence which codes for additional aminoacids, such as those which provide additional functionalities.

[0072] Thus, the sequence encoding the polypeptide may be fused to amarker sequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described by Gentzand colleagues (Proc. Natl. Acad. Sci. USA 86:821-824 (1989)), forinstance, hexa-histidine provides for convenient purification of thefusion protein. The “HA” tag is another peptide useful for purificationwhich corresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson and coworkers (Cell 37:767(1984)). As discussed below, other such fusion proteins include thefollistatin-3 fused to Fc at the N- or C-terminus.

[0073] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the follistatin-3 protein. Variants may occurnaturally, such as a natural allelic variant. By an “allelic variant” isintended one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985)). Non-naturally occurring variants may beproduced using art-known mutagenesis techniques.

[0074] Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the follistatin-3 protein or portionsthereof. Also especially preferred in this regard are conservativesubstitutions.

[0075] Most highly preferred are nucleic acid molecules encoding themature protein having the amino acid sequence shown in SEQ ID NO:2 orthe mature follistatin-3 amino acid sequence encoded by the depositedcDNA clone.

[0076] Further embodiments include an isolated nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence at least 90%identical, and more preferably at least 95%, 96%, 97%, 98% or 99%identical to a polynucleotide selected from the group consisting of: (a)a nucleotide sequence encoding the follistatin-3 polypeptide having thecomplete amino acid sequence in SEQ ID NO:2 (i.e., positions −26 to 237of SEQ ID NO:2); (b) a nucleotide sequence encoding the follistatin-3polypeptide having the complete amino acid sequence in SEQ ID NO:2excepting the N-terminal methionine (i.e., positions −25 to 237 of SEQID NO:2); (c) a nucleotide sequence encoding the predicted maturefollistatin-3 polypeptide having the amino acid sequence at positions 1to 237 in SEQ ID NO:2; (d) a nucleotide sequence encoding thefollistatin-3 polypeptide having the complete amino acid sequenceencoded by the cDNA clone contained in ATCC® Deposit No. 209199; (e) anucleotide sequence encoding the follistatin-3 polypeptide having thecomplete amino acid sequence excepting the N-terminal methionine encodedby the cDNA clone contained in ATCC® Deposit No. 209199; (f) anucleotide sequence encoding the mature follistatin-3 polypeptide havingthe amino acid sequence encoded by the cDNA clone contained in ATCC®Deposit No. 209199; and (g) a nucleotide sequence complementary to anyof the nucleotide sequences in (a), (b), (c), (d), (e) or (f) above.

[0077] Further embodiments of the invention include isolated nucleicacid molecules that comprise a polynucleotide having a nucleotidesequence at least 90% identical, and more preferably at least 95%, 96%,97%, 98% or 99% identical, to any of the nucleotide sequences in (a),(b), (c), (d), (e), (f) or (g), above, or a polynucleotide whichhybridizes under stringent hybridization conditions to a polynucleotidein (a), (b), (c), (d), (e), (f) or (g), above. This polynucleotide whichhybridizes does not hybridize under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues. An additional nucleic acid embodiment ofthe invention relates to an isolated nucleic acid molecule comprising apolynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a follistatin-3 polypeptide having an aminoacid sequence in (a), (b), (c), (d), (e) or (f), above. A furthernucleic acid embodiment of the invention relates to an isolated nucleicacid molecule comprising a polynucleotide which encodes the amino acidsequence of a follistatin-3 polypeptide having an amino acid sequencewhich contains at least one conservative amino acid substitution, butnot more than 50 conservative amino acid substitutions, even morepreferably, not more than 40 conservative amino acid substitutions,still more preferably not more than 30 conservative amino acidsubstitutions, and still even more preferably not more than 20conservative amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a polynucleotidewhich encodes the amino acid sequence of a follistatin-3 polypeptide tohave an amino acid sequence which contains not more than 7-10, 5-10,3-7, 3-5, 2-5, 1-5, 1-3, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservativeamino acid substitutions.

[0078] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of follistatin-3 polypeptides or peptides by recombinanttechniques.

[0079] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding afollistatin-3 polypeptide is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequences encoding thefollistatin-3 polypeptides. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5′ or 3′ terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

[0080] As a practical matter, whether any particular nucleic acidmolecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in FIGS. 1A, 1B, and 1C, or tothe nucleotides sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman to find the best segment of homology between two sequences(Advances in Applied Mathematics 2:482-489 (1981)). When using Bestfitor any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed. A preferred method for determing the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)). In asequence alignment the query and subject sequences are both DNAsequences. An RNA sequence can be compared by converting U's to T's. Theresult of said global sequence alignment is in percent identity.Preferred parameters used in a FASTDB alignment of DNA sequences tocalculate percent identiy are: Matrix=Unitary, k-tuple=4, MismatchPenalty=1, Joining Penalty=30, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or thelength of the subject nucleotide sequence, whichever is shorter.

[0081] If the subject sequence is shorter than the query sequencebecause of 5′ or 3′ deletions, not because of internal deletions, amanual correction must be made to the results. This is becuase theFASTDB program does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.Whether a nucleotide is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thiscorrected score is what is used for the purposes of the presentinvention. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score.

[0082] For example, a 90 base subject sequence is aligned to a 100 basequery sequence to determine percent identity. The deletions occur at the5′ end of the subject sequence and therefore, the FASTDB alignment doesnot show a matched/alignement of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequnce aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

[0083] The present application is directed to nucleic acid molecules atleast 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) or to the nucleicacid sequence of the deposited cDNA, irrespective of whether they encodea polypeptide having follistatin-3 activity. This is because even wherea particular nucleic acid molecule does not encode a polypeptide havingfollistatin-3 activity, one of skill in the art would still know how touse the nucleic acid molecule, for instance, as a hybridization probe ora polymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving follistatin-3 activity include, inter alia, (1) isolating thefollistatin-3 gene or allelic variants thereof in a cDNA library; (2) insitu hybridization (e.g., “FISH”) to metaphase chromosomal spreads toprovide precise chromosomal location of the follistatin-3 gene, asdescribed by Verma and colleagues (Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988)); and Northern Blot analysisfor detecting follistatin-3 mRNA expression in specific tissues.

[0084] Preferred, however, are nucleic acid molecules having sequencesat least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) or to the nucleicacid sequence of the deposited cDNA which do, in fact, encode apolypeptide having follistatin-3 protein activity. By “a polypeptidehaving follistatin-3 activity” is intended polypeptides exhibitingactivity similar, but not necessarily identical, to an activity of themature follistatin-3 protein of the invention, as measured in aparticular biological assay. For example, the follistatin-3 protein ofthe present invention inhibits the binding of activin to the activinreceptor. An activin receptor-binding inhibition assay is described byHashimoto and colleagues (J. Biol. Chem. 272:13835-13842 (1997)).Briefly, the assay involves culturing rat pituitary cells (5×10⁵ cells)in 24-well plates in the presence of [¹²⁵I]-activin A (40 ng/mL; activinA is labeled using the chloramine-T method as described by Hasegawa andcoworkers (Endocrinol. Japan 33:645-654 (1986)) and follistatin-3 or amutein thereof (200 ng/mL). A baseline of activin-binding is determinedby affinity cross-linking [¹²⁵I]-activin A to the pituitary cells usingthe bifunctional chemical cross-linker disuccinimidyl suberate (DSS) inthe absence of follistatin-3. Cross-linking is achieved by washing cellsonce with binding buffer (DMEM containing 25 mM HEPES (pH 7.4) and 0.2%bovine serum albumen) and incubating on ice for 2 h with 40 ng/mL[¹²⁵I]-activin A in the binding buffer. Following incubation, cells arewashed 3 times with ice-cold PBS and incubated in PBS containing 1 mMDSS for 20 min on ice. The reaction is then quenched with PBS. The cellsare removed from the culture dish by scraping, rinsed with a Trissolution (20 mM Tris-HCl (pH 7.2) containing 2 mM EDTA, 5 mMbenzamidine, 2 mM phenylmethylsulfonyl fluoride (PMSF), 2 mMN-ethylaleimide, and 2 mM diisopropyl fluorophosphate), centrifuged, andresuspended in solubilization buffer (50 mM Tris-HCl (pH 7.2) containing150 mM NaCl, 2 mM EDTA, 5 mM benzamidine, 2 mM PMSF, 2 mMN-ethylaleimide, 2 mM diisopropyl fluorophosphate, 1% Triton X-100, and10% glycerol), and stirred gently on ice for 1 h. The cell lysates areintroduced into 2% SDS and boiled at 100 C for 10 min. The resultingaffinity-labeled lysates are then subject to SDS-PAGE (7.5 or 8% gels).Following SDS-PAGE, gels are fixed, stained with 0.25% CoomassieBrilliant Blue R-250, destained, air-dried, and then visualized byautoradiography. Inhibition of activin binding of the activin receptoris analyzed in samples with which follistatin-3 or a mutein thereof (200ng/mL) are incubated with labeled activin in the binding bufferincubation described above. The degree to which the formation ofaffinity cross-linked activin/activin receptor complexes is decreasedcorrelates with the ability of follistatin-3 or a mutein thereof to bindto labeled activin protein. As such, the relative binding affinity ofactivin for its receptor versus follistatin-3 or a mutein thereof can bequantitated. Such activity is useful for regulating the effective amountof activin present in a given system.

[0085] Follistatin-3 protein binds to activin in a dose-dependent mannerin the above-described assay. Thus, “a polypeptide having follistatin-3protein activity” includes polypeptides that also exhibit any of thesame binding activities in the above-described assays in adose-dependent manner. Although the degree of dose-dependent activityneed not be identical to that of the follistatin-3 protein, preferably,“a polypeptide having follistatin-3 protein activity” will exhibitsubstantially similar dose-dependence in a given activity as compared tothe follistatin-3 protein (i.e., the candidate polypeptide will exhibitgreater activity or not more than about 25-fold less and, preferably,not more than about tenfold less activity relative to the referencefollistatin-3 protein).

[0086] Like follistatin-1, follistatin-3 inhibits the secretion of FSH.An assay for measuring the suppression of spontaneous FSH release fromprimary cultured rat pituitary cells is well known in the art (Hasegawa,Y., et al., Endocrinol. Jpn. 33:645-654 (1986)). Briefly, freshlyisolated pituitary cells are suspended in DMEM containing gentamicin (35μg/mL), fungizone (1 μg/mL), 0.05% glutamine, 0.1% sodium bicarbonate,10% horse serum, and 2.5% fetal bovine serum at a density of 3×10⁵cells/mL, and plated in 96-well culture plates (6×10⁴ cells/0.2mL/well). Various amounts (0.1-100 ng/mL) of follistatin-3 are thenadded to the culture medium. After culturing for 3 days at 37 C (5%CO₂), cultured media are assayed for quantity of secreted FSH by adouble antibody RIA method using an RIA kit and plotted as FSH Secreted(ng/mL/72 h) versus Protein Added (ng/mL).

[0087] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 90%, 95%, 96%,97%, 98%, or 99% identical to the nucleic acid sequence of the depositedcDNA or the nucleic acid sequence shown in FIGS. 1A, 1B, and 1C (SEQ IDNO:1) will encode a polypeptide “having follistatin-3 protein activity.”In fact, since degenerate variants of these nucleotide sequences allencode the same polypeptide, this will be clear to the skilled artisaneven without performing the above described comparison assay. It will befurther recognized in the art that, for such nucleic acid molecules thatare not degenerate variants, a reasonable number will also encode apolypeptide having follistatin-3 protein activity. This is because theskilled artisan is fully aware of amino acid substitutions that areeither less likely or not likely to significantly effect proteinfunction (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid), as further described below.

[0088] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof follistatin-3 polypeptides or fragments thereof by recombinanttechniques. The vector may be, for example, a phage, plasmid, viral orretroviral vector. Retroviral vectors may be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells.

[0089] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0090] The DNA insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

[0091] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase, G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

[0092] Vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9 (QIAGEN, Inc., supra); pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Among preferredeukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1, and pSG(Stratagene); and pSVK3, pBPV, pMSG and pSVL (Pharmacia). Other suitablevectors will be readily apparent to the skilled artisan.

[0093] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals (for example, Davis, et al., Basic Methods InMolecular Biology (1986)).

[0094] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to stabilize andpurify proteins. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5(Bennett, D., et al., J. Molecular Recognition 8:52-58 (1995); Johanson,K., et al., J. Biol. Chem. 270:9459-9471 (1995)).

[0095] The follistatin-3 protein can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Polypeptides of the present invention include: products purified fromnatural sources, including bodily fluids, tissues and cells, whetherdirectly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the polypeptides of thepresent invention may be glycosylated or may be non-glycosylated. Inaddition, polypeptides of the invention may also include an initialmodified methionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

Polypeptides and Fragments

[0096] The invention further provides an isolated follistatin-3polypeptide having the amino acid sequence encoded by the depositedcDNA, or the amino acid sequence in SEQ ID NO:2, or a peptide orpolypeptide comprising a portion of the above polypeptides.

[0097] To improve or alter the characteristics of follistatin-3polypeptides, protein engineering may be employed. Recombinant DNAtechnology known to those skilled in the art can be used to create novelmutant proteins or muteins including single or multiple amino acidsubstitutions, deletions, additions or fusion proteins. Such modifiedpolypeptides can show, e.g., enhanced activity or increased stability.In addition, they may be purified in higher yields and show bettersolubility than the corresponding natural polypeptide, at least undercertain purification and storage conditions.

[0098] For instance, for many proteins, including the extracellulardomain of a membrane associated protein or the mature form(s) of asecreted protein, it is known in the art that one or more amino acidsmay be deleted from the N-terminus or C-terminus without substantialloss of biological function. For instance, Ron and colleagues (J. Biol.Chem., 268:2984-2988 (1993)) reported modified KGF proteins that hadheparin binding activity even if 3, 8, or 27 N-terminal amino acidresidues were missing. In the present case, since the protein of theinvention is a member of the inhibin-related polypeptide family,deletions of N-terminal amino acids up to the cysteine at position 12 ofSEQ ID NO:2 may retain some biological activity such as binding activinor an activin-like molecule. Polypeptides having further N-terminaldeletions including the cysteine-12 residue in SEQ ID NO:2 would not beexpected to retain such biological activities because it is known thatthis residue is likely required for forming a disulfide bridge toprovide structural stability which is needed for protein-proteininteraction and is in the beginning of the conserved domain required forbiological activities.

[0099] However, even if deletion of one or more amino acids from theN-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other biological activities maystill be retained. Thus, the ability of the shortened protein to induceand/or bind to antibodies which recognize the complete or mature of theprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

[0100] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of the aminoacid sequence of the follistatin-3 shown in SEQ ID NO:2, up to thecysteine residue at position number 12, and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides comprising the amino acid sequence of residues n¹-237 ofSEQ ID NO:2, where n is an integer in the range of −26-12, and 12 is theposition of the first residue from the N-terminus of the completefollistatin-3 polypeptide (shown in SEQ ID NO:2) believed to be requiredfor activin-binding or activin-like protein-binding activity of thefollistatin-3 protein.

[0101] More in particular, the invention provides polynucleotidesencoding polypeptides having the amino acid sequence of residues of−26-237, −25-237, −24-237, −23-237, −22-237, −21-237, −20-237, −19-237,−18-237, −17-237, −16-237, −15-237, −14-237, −13-237, −12-237, −10-237,−9-237, −8-237, −7-237, −6-237, −5-237, −4-237, −3-237, −2-237, −1-237,1-237, 2-237, 3-237, 4-237, 5-237, 6-237, 7-237, 8-237, 9-237, 10-237,11-237, and 12-237 of SEQ ID NO:2. Polypeptides encoded by thesepolynucleotides also are provided. The present invention is alsodirected to nucleic acid molecules comprising, or alternatively,consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%,94%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequencesencoding the follistatin-3 polypeptides described above, and thepolypeptides encoded thereby. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence, and the polypeptides encoded thereby.

[0102] Similarly, many examples of biologically functional C-terminaldeletion muteins are known. For instance, Interferon gamma shows up toten times higher activities by deleting 8-10 amino acid residues fromthe carboxy terminus of the protein (Dobeli, et al., J. Biotechnology7:199-216 (1988)). In the present case, since the protein of theinvention is a member of the activin-related polypeptide family,deletions of C-terminal amino acids up to the cysteine at position 217of SEQ ID NO:2 may retain some biological activity such as bindingactivin or an activin-like molecule. Polypeptides having furtherC-terminal deletions including the cysteine residue at position 217 ofSEQ ID NO:2 would not be expected to retain such biological activitiesbecause it is known that this residue is likely required for forming adisulfide bridge to provide structural stability which is needed forprotein-protein interactions and is the beginning of the conserveddomain required for biological activities.

[0103] However, even if deletion of one or more amino acids from theC-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other biological activities maystill be retained. Thus, the ability of the shortened protein to induceand/or bind to antibodies which recognize the complete or mature form ofthe protein generally will be retained when less than the majority ofthe residues of the complete or mature form of the protein are removedfrom the C-terminus. Whether a particular polypeptide lacking C-terminalresidues of a complete protein retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art.

[0104] Accordingly, the present invention further provides polypeptideshaving one or more residues from the carboxy terminus of the amino acidsequence of the follistatin-3 shown in SEQ ID NO:2, up to the cysteineresidue at position 217 of SEQ ID NO:2, and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides having the amino acid sequence of residues −26-m¹ of theamino acid sequence in SEQ ID NO:2, where m¹ is any integer in the rangeof 217 to 237, and residue 217 is the position of the first residue fromthe C-terminus of the complete follistatin-3 polypeptide (shown in SEQID NO:2) believed to be required for the activin-binding or activin-likeprotein-binding of the follistatin-3 protein.

[0105] More in particular, the invention provides polynucleotidesencoding polypeptides having the amino acid sequence of residues−26-217, −26-218, −26-219, −26-220, −26-221, −26-222, −26-223, −26-224,−26-225, −26-226, −26-227, −26-228, −26-229, −26-230, −26-231, −26-232,−26-233, −26-234, −26-235, −26-236, and −26-237 of SEQ ID NO:2.Polypeptides encoded by these polynucleotides also are provided. Thepresent invention is also directed to nucleic acid molecules comprising,or alternatively, consisting of, a polynucleotide sequence at least 80%,85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to thepolynucleotide sequences encoding the follistatin-3 polypeptidesdescribed above, and the polypeptides encoded thereby. The presentinvention also encompasses the above polynucleotide sequences fused to aheterologous polynucleotide sequence, and the polypeptides encodedthereby.

[0106] The invention also provides polypeptides having one or more aminoacids deleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues n¹-m¹ of SEQ ID NO:2, where n¹and m¹ are integers as described above.

[0107] Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete follistatin-3 amino acidsequence encoded by the cDNA clone contained in ATCC® Deposit No.209199, where this portion excludes from 1 to about 37 amino acids fromthe amino terminus of the complete amino acid sequence encoded by thecDNA clone contained in ATCC® Deposit No. 209199, or from 1 to about 20amino acids from the carboxy terminus, or any combination of the aboveamino terminal and carboxy terminal deletions, of the complete aminoacid sequence encoded by the cDNA clone contained in ATCC® Deposit No.209199. Polynucleotides encoding all of the above deletion mutantpolypeptide forms also are provided.

[0108] As mentioned above, even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other biological activitiesmay still be retained. Thus, the ability of the shortened follistatin-3mutein to induce and/or bind to antibodies which recognize the completeor mature of the protein generally will be retained when less than themajority of the residues of the complete or mature protein are removedfrom the N-terminus. Whether a particular polypeptide lacking N-terminalresidues of a complete protein retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a follistatin-3 mutein with alarge number of deleted N-terminal amino acid residues may retain somebiological or immungenic activities. In fact, peptides composed of asfew as six follistatin-3 amino acid residues may often evoke an immuneresponse.

[0109] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of thefollistatin-3 amino acid sequence shown in SEQ ID NO:2, up to theglutamic acid residue at position number 258 and polynucleotidesencoding such polypeptides. In particular, the present inventionprovides polypeptides comprising the amino acid sequence of residuesn²-263 of FIG. 1A (SEQ ID NO:2), where n² is an integer in the range of2 to 258, and 259 is the position of the first residue from theN-terminus of the complete follistatin-3 polypeptide believed to berequired for at least immunogenic activity of the follistatin-3 protein.

[0110] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of residues of R-2 to V-263; P-3 to V-263; G-4 toV-263; A-5 to V-263; P-6 to V-263; G-7 to V-263; P-8 to V-263; L-9 toV-263; W-10 to V-263; P-11 to V-263; L-12 to V-263; P-13 to V-263; W-14to V-263; G-15 to V-263; A-16 to V-263; L-17 to V-263; A-18 to V-263;W-19 to V-263; A-20 to V-263; V-21 to V-263; G-22 to V-263; F-23 toV-263; V-24 to V-263; S-25 to V-263; S-26 to V-263; M-27 to V-263; G-28to V-263; S-29 to V-263; G-30 to V-263; N-31 to V-263; P-32 to V-263;A-33 to V-263; P-34 to V-263; G-35 to V-263; G-36 to V-263; V-37 toV-263; C-38 to V-263; W-39 to V-263; L-40 to V-263; Q-41 to V-263; Q-42to V-263; G-43 to V-263; Q-44 to V-263; E-45 to V-263; A-46 to V-263;T-47 to V-263; C-48 to V-263; S-49 to V-263; L-50 to V-263; V-51 toV-263; L-52 to V-263; Q-53 to V-263; T-54 to V-263; D-55 to V-263; V-56to V-263; T-57 to V-263; R-58 to V-263; A-59 to V-263; E-60 to V-263;C-61 to V-263; C-62 to V-263; A-63 to V-263; S-64 to V-263; G-65 toV-263; N-66 to V-263; 1-67 to V-263; D-68 to V-263; T-69 to V-263; A-70to V-263; W-71 to V-263; S-72 to V-263; N-73 to V-263; L-74 to V-263;T-75 to V-263; H-76 to V-263; P-77 to V-263; G-78 to V-263; N-79 toV-263; K-80 to V-263; I-81 to V-263; N-82 to V-263; L-83 to V-263; L-84to V-263; G-85 to V-263; F-86 to V-263; L-87 to V-263; G-88 to V-263;L-89 to V-263; V-90 to V-263; H-91 to V-263; C-92 to V-263; L-93 toV-263; P-94 to V-263; C-95 to V-263; K-96 to V-263; D-97 to V-263; S-98to V-263; C-99 to V-263; D-100 to V-263; G-101 to V-263; V-102 to V-263;E-103 to V-263; C-104 to V-263; G-105 to V-263; P-106 to V-263; G-107 toV-263; K-108 to V-263; A-109 to V-263; C-I 10 to V-263; R-111 to V-263;M-112 to V-263; L-113 to V-263; G-114 to V-263; G-115 to V-263; R-116 toV-263; P-117 to V-263; R-118 to V-263; C-119 to V-263; E-120 to V-263;C-121 to V-263; A-122 to V-263; P-123 to V-263; D-124 to V-263; C-125 toV-263; S-126 to V-263; G-127 to V-263; L-128 to V-263; P-129 to V-263;A-130 to V-263; R-131 to V-263; L-132 to V-263; Q-133 to V-263; V-134 toV-263; C-135 to V-263; G-136 to V-263; S-137 to V-263; D-138 to V-263;G-139 to V-263; A-140 to V-263; T-141 to V-263; Y-142 to V-263; R-143 toV-263; D-144 to V-263; E-145 to V-263; C-146 to V-263; E-147 to V-263;L-148 to V-263; R-149 to V-263; A-150 to V-263; A-151 to V-263; R-152 toV-263; C-153 to V-263; R-154 to V-263; G-155 to V-263; H-156 to V-263;P-157 to V-263; D-158 to V-263; L-159 to V-263; S-160 to V-263; V-161 toV-263; M-162 to V-263; Y-163 to V-263; R-164 to V-263; G-165 to V-263;R-166 to V-263; C-167 to V-263; R-168 to V-263; K-169 to V-263; S-170 toV-263; C-171 to V-263; E-172 to V-263; H-173 to V-263; V-174 to V-263;V-175 to V-263; C-176 to V-263; P-177 to V-263; R-178 to V-263; P-179 toV-263; Q-180 to V-263; S-181 to V-263; C-182 to V-263; V-183 to V-263;V-184 to V-263; D-185 to V-263; Q-186 to V-263; T-187 to V-263; G-188 toV-263; S-189 to V-263; A-190 to V-263; H-191 to V-263; C-192 to V-263;V-193 to V-263; V-194 to V-263; C-195 to V-263; R-196 to V-263; A-197 toV-263; A-198 to V-263; P-199 to V-263; C-200 to V-263; P-201 to V-263;V-202 to V-263; P-203 to V-263; S-204 to V-263; S-205 to V-263; P-206 toV-263; G-207 to V-263; Q-208 to V-263; E-209 to V-263; L-210 to V-263;C-211 to V-263; G-212 to V-263; N-213 to V-263; N-214 to V-263; N-215 toV-263; V-216 to V-263; T-217 to V-263; Y-218 to V-263; I-219 to V-263;S-220 to V-263; S-221 to V-263; C-222 to V-263; H-223 to V-263; M-224 toV-263; R-225 to V-263; Q-226 to V-263; A-227 to V-263; T-228 to V-263;C-229 to V-263; F-230 to V-263; L-231 to V-263; G-232 to V-263; R-233 toV-263; S-234 to V-263; I-235 to V-263; G-236 to V-263; V-237 to V-263;R-238 to V-263; H-239 to V-263; A-240 to V-263; G-241 to V-263; S-242 toV-263; C-243 to V-263; A-244 to V-263; G-245 to V-263; T-246 to V-263;P-247 to V-263; E-248 to V-263; E-249 to V-263; P-250 to V-263; P-251 toV-263; G-252 to V-263; G-253 to V-263; E-254 to V-263; S-255 to V-263;A-256 to V-263; E-257 to V-263; and E-258 to V-263 of the follistatin-3amino acid sequence shown in FIG. 1A (which is identical to the sequenceshown as SEQ ID NO:2, with the exception that the amino acid residues inFIG. 1A are numbered consecutively from 1 through 263 from theN-terminus to the C-terminus, while the amino acid residues in SEQ IDNO:2 are numbered consecutively from −26 through 237 to reflect theposition of the predicted signal peptide). Polypeptides encoded by thesepolynucleotides also are provided. The present invention is alsodirected to nucleic acid molecules comprising, or alternatively,consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%,94%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequencesencoding the follistatin-3 polypeptides described above, and thepolypeptides encoded thereby. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence, and the polypeptides encoded thereby.

[0111] Also as mentioned above, even if deletion of one or more aminoacids from the C-terminus of a protein results in modification of lossof one or more biological functions of the protein, other biologicalactivities may still be retained. Thus, the ability of the shortenedfollistatin-3 mutein to induce and/or bind to antibodies which recognizethe complete or mature of the protein generally will be retained whenless than the majority of the residues of the complete or mature proteinare removed from the C-terminus. Whether a particular polypeptidelacking C-terminal residues of a complete protein retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thata follistatin-3 mutein with a large number of deleted C-terminal aminoacid residues may retain some biological or immungenic activities. Infact, peptides composed of as few as six follistatin-3 amino acidresidues may often evoke an immune response.

[0112] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the carboxy terminus of theamino acid sequence of the follistatin-3 shown in SEQ ID NO:2, up to theproline residue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m² of SEQ ID NO:2,where m² is an integer in the range of 6 to 262, and 6 is the positionof the first residue from the C-terminus of the complete follistatin-3polypeptide believed to be required for at least immunogenic activity ofthe follistatin-3 protein.

[0113] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of residues M-1 to F-262; M-1 to N-261; M-1 toE-260; M-1 to E-259; M-1 to E-258; M-1 to E-257; M-1 to A-256; M-1 toS-255; M-1 to E-254; M-1 to G-253; M-1 to G-252; M-1 to P-251; M-1 toP-250; M-1 to E-249; M-1 to E-248; M-1 to P-247; M-1 to T-246; M-1 toG-245; M-1 to A-244; M-1 to C-243; M-1 to S-242; M-1 to G-241; M-1 toA-240; M-1 to H-239; M-1 to R-238; M-1 to V-237; M-1 to G-236; M-1 to1-235; M-1 to S-234; M-1 to R-233; M-1 to G-232; M-1 to L-231; M-1 toF-230; M-1 to C-229; M-1 to T-228; M-1 to A-227; M-1 to Q-226; M-1 toR-225; M-1 to M-224; M-1 to H-223; M-1 to C-222; M-1 to S-221; M-1 toS-220; M-1 to 1-219; M-1 to Y-218; M-1 to T-217; M-1 to V-216; M-1 toN-215; M-1 to N-214; M-1 to N-213; M-1 to G-212; M-1 to C-211; M-1 toL-210; M-1 to E-209; M-1 to Q-208; M-1 to G-207; M-1 to P-206; M-1 toS-205; M-1 to S-204; M-1 to P-203; M-1 to V-202; M-1 to P-201; M-1 toC-200; M-1 to P-199; M-1 to A-198; M-1 to A-197; M-1 to R-196; M-1 toC-195; M-1 to V-194; M-1 to V-193; M-1 to C-192; M-1 to H-191; M-1 toA-190; M-1 to S-189; M-1 to G-188; M-1 to T-187; M-1 to Q-186; M-1 toD-185; M-1 to V-184; M-1 to V-183; M-1 to C-182; M-1 to S-181; M-1 toQ-180; M-1 to P-179; M-1 to R-178; M-1 to P-177; M-1 to C-176; M-1 toV-175; M-1 to V-174; M-1 to H-173; M-1 to E-172; M-1 to C-171; M-1 toS-170; M-1 to K-169; M-1 to R-168; M-1 to C-167; M-1 to R-166; M-1 toG-165; M-1 to R-164; M-1 to Y-163; M-1 to M-162; M-1 to V-161; M-1 toS-160; M-1 to L-159; M-1 to D-158; M-1 to P-157; M-1 to H-156; M-1 toG-155; M-1 to R-154; M-1 to C-153; M-1 to R-152; M-1 to A-151; M-1 toA-150; M-1 to R-149; M-1 to L-148; M-1 to E-147; M-1 to C-146; M-1 toE-145; M-1 to D-144; M-1 to R-143; M-1 to Y-142; M-1 to T-141; M-1 toA-140; M-1 to G-139; M-1 to D-138; M-1 to S-137; M-1 to G-136; M-1 toC-135; M-1 to V-134; M-1 to Q-133; M-1 to L-132; M-1 to R-131; M-1 toA-130; M-1 to P-129; M-1 to L-128; M-1 to G-127; M-1 to S-126; M-1 toC-125; M-1 to D-124; M-1 to P-123; M-1 to A-122; M-1 to C-121; M-1 toE-120; M-1 to C-119; M-1 to R-118; M-1 to P-117; M-1 to R-116; M-1 toG-115; M-1 to G-114; M-1 to L-113; M-1 to M-112; M-1 to R-111; M-1 toC-110; M-1 to A-109; M-1 to K-108; M-1 to G-107; M-1 to P-106; M-1 toG-105; M-1 to C-104; M-1 to E-103; M-1 to V-102; M-1 to G-101; M-1 toD-100; M-1 to C-99; M-1 to S-98; M-1 to D-97; M-1 to K-96; M-1 to C-95;M-1 to P-94; M-1 to L-93; M-1 to C-92; M-1 to H-91; M-1 to V-90; M-1 toL-89; M-1 to G-88; M-1 to L-87; M-1 to F-86; M-1 to G-85; M-1 to L-84;M-1 to L-83; M-1 to N-82; M-1 to 1-81; M-1 to K-80; M-1 to N-79; M-1 toG-78; M-1 to P-77; M-1 to H-76; M-1 to T-75; M-1 to L-74; M-1 to N-73;M-1 to S-72; M-1 to W-71; M-1 to A-70; M-1 to T-69; M-1 to D-68; M-1 to1-67; M-1 to N-66; M-1 to G-65; M-1 to S-64; M-1 to A-63; M-1 to C-62;M-1 to C-61; M-1 to E-60; M-1 to A-59; M-1 to R-58; M-1 to T-57; M-1 toV-56; M-1 to D-55; M-1 to T-54; M-1 to Q-53; M-1 to L-52; M-1 to V-51;M-1 to L-50; M-1 to S-49; M-1 to C-48; M-1 to T-47; M-1 to A-46; M-1 toE-45; M-1 to Q-44; M-1 to G-43; M-1 to Q-42; M-1 to Q-41; M-1 to L-40;M-1 to W-39; M-1 to C-38; M-1 to V-37; M-1 to G-36; M-1 to G-35; M-1 toP-34; M-1 to A-33; M-1 to P-32; M-1 to N-31; M-1 to G-30; M-1 to S-29;M-1 to G-28; M-1 to M-27; M-1 to S-26; M-1 to S-25; M-1 to V-24; M-1 toF-23; M-1 to G-22; M-1 to V-21; M-1 to A-20; M-1 to W-19; M-1 to A-18;M-1 to L-17; M-1 to A-16; M-1 to G-15; M-1 to W-14; M-1 to P-13; M-1 toL-12; M-1 to P-11; M-1 to W-10; M-1 to L-9; M-1 to P-8; M-1 to G-7; M-1to P-6 of the sequence of the follistatin-3 sequence shown in FIG. 1A(which is identical to the sequence shown as SEQ ID NO:2, with theexception that the amino acid residues in FIG. 1A are numberedconsecutively from 1 through 263 from the N-terminus to the C-terminus,while the amino acid residues in SEQ ID NO:2 are numbered consecutivelyfrom −26 through 237 to reflect the position of the predicted signalpeptide). Polypeptides encoded by these polynucleotides also areprovided. The present invention is also directed to nucleic acidmolecules comprising, or alternatively, consisting of, a polynucleotidesequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%identical to the polynucleotide sequences encoding the follistatin-3polypeptides described above, and the polypeptides encoded thereby. Thepresent invention also encompasses the above polynucleotide sequencesfused to a heterologous polynucleotide sequence, and the polypeptidesencoded thereby.

[0114] The invention also provides polypeptides having one or more aminoacids deleted from both the amino and the carboxyl termini of afollistatin-3 polypeptide, which may be described generally as havingresidues n²-m² of FIG. 1A (SEQ ID NO:2), where n² and m² are integers asdescribed above.

[0115] In addition to terminal deletion forms of the protein discussedabove, it also will be recognized by one of ordinary skill in the artthat some amino acid sequences of the follistatin-3 polypeptide can bevaried without significant effect of the structure or function of theprotein. If such differences in sequence are contemplated, it should beremembered that there will be critical areas on the protein whichdetermine activity.

[0116] Thus, the invention further includes variations of thefollistatin-3 polypeptide which show substantial follistatin-3polypeptide activity or which include regions of follistatin-3 proteinsuch as the protein portions discussed below. Such mutants includedeletions, insertions, inversions, repeats, and type substitutionsselected according to general rules known in the art so as have littleeffect on activity. For example, guidance concerning how to makephenotypically silent amino acid substitutions is provided wherein theauthors indicate that there are two main approaches for studying thetolerance of an amino acid sequence to change (Bowie, J. U., et al.,Science 247:1306-1310 (1990)). The first method relies on the process ofevolution, in which mutations are either accepted or rejected by naturalselection. The second approach uses genetic engineering to introduceamino acid changes at specific positions of a cloned gene and selectionsor screens to identify sequences that maintain functionality.

[0117] As the authors state, these studies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive ata certain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described by Bowie and coworkers (supra) and thereferences cited therein. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

[0118] Thus, the fragment, derivative or analog of the polypeptide ofSEQ ID NO:2, or that encoded by the deposited cDNA, may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the above form of the polypeptide, such as an IgG Fc fusionregion peptide or leader or secretory sequence or a sequence which isemployed for purification of the above form of the polypeptide or aproprotein sequence. Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein.

[0119] Thus, the follistatin-3 of the present invention may include oneor more amino acid substitutions, deletions or additions, either fromnatural mutations or human manipulation. As indicated, changes arepreferably of a minor nature, such as conservative amino acidsubstitutions that do not significantly affect the folding or activityof the protein (see Table II). TABLE II Conservative Amino AcidSubstitutions. Aromatic Phenylalanine Tryptophan Tyrosine HydrophobicLeucine Isoleucine Valine Polar Glutamine Asparagine Basic ArginineLysine Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine SerineThreonine Methionine Glycine

[0120] Embodiments of the invention are directed to polypeptides whichcomprise the amino acid sequence of a follistatin-3 polypeptidedescrubed hereub, but having an amino acid sequence which contains atleast one conservative amino acid substitution, but not more than 50conservative amino acid substitutions, even more preferably, not morethan 40 conservative amino acid substitutions, still more preferably,not more than 30 conservative amino acid substitutions, and still evenmore preferably, not more than 20 conservative amino acid substitutions,when compared with the follistatin-3 polynucleotide sequence describedherein. Of course, in order of ever-increasing preference, it is highlypreferable for a peptide or polypeptide to have an amino acid sequencewhich comprises the amino acid sequence of a follistatin-3 polypeptide,which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 conservative amino acid substitutions.

[0121] In further specific embodiments, the number of substitutions,additions or deletions in the amino acid sequence of FIGS. 1A, 1B, and1C (SEQ ID NO:2), a polypeptide sequence encoded by the depositedclones, and/or any of the polypeptide fragments described herein is 75,70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or150-50, 100-50, 50-20, 30-20, 20-15, 20-10, 15-10, 10-1, 5-10, 1-5, 1-3or 1-2.

[0122] To improve or alter the characteristics of follistatin-3polypeptides, protein engineering may be employed. Recombinant DNAtechnology known to those skilled in the art can be used to create novelmutant proteins or muteins including single or multiple amino acidsubstitutions, deletions, additions or fusion proteins. Such modifiedpolypeptides can show, e.g., enhanced activity or increased stability.In addition, they may be purified in higher yields and show bettersolubility than the corresponding natural polypeptide, at least undercertain purification and storage conditions.

[0123] Thus, the invention also encompasses follistatin-3 derivativesand analogs that have one or more amino acid residues deleted, added, orsubstituted to generate follistatin-3 polypeptides that are bettersuited for expression, scale up, etc., in the host cells chosen. Forexample, cysteine residues can be deleted or substituted with anotheramino acid residue in order to eliminate disulfide bridges; N-linkedglycosylation sites can be altered or eliminated to acheive, forexample, expression of a homogeneous product that is more easilyrecovered and purified from yeast hosts which are known tohyperglycosylate N-linked sites. To this end, a variety of amino acidsubstitutions at one or both of the first or third amino acid positionson any one or more of the glycosylation recognitions sequences in thefollistatin-3 polypeptides of the invention, and/or an amino aciddeletion at the second position of any one or more such recognitionsequences will prevent glycosylation of the follistatin-3 polypeptide atthe modified tripeptide sequence (see, e.g., Miyajima, A., et al., EMBOJ. 5(6):1193-1197 (1986)).

[0124] Amino acids in the follistatin-3 protein of the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such as receptor binding or in vitro proliferativeactivity.

[0125] Of special interest are substitutions of charged amino acids withother charged or neutral amino acids which may produce proteins withhighly desirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard, et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins, et al., Diabetes 36:838-845 (1987); Cleland, et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993)).

[0126] A mutational analysis of the two N-linked glycosylation sites(Asn-95 and Asn-259) of follistatin-1 was conducted by Inouye andcolleagues (Biochem. Biophys. Res. Comm. 179:352-358 (1991)). Asdescribed in the analysis, disruption of either or both of the N-linkedglycosylation sites (by mutation of Thr-97 and Thr-261 to alanine) hadno discernable effect on activin-binding and FSH secretion. However,results of the same study suggest that insertion of two amino acidresidues (lysine and leucine) between residues Asn-2 and Cys-3 offollistatin-1 completely abolishes its inhibitory activity on FSHsecretion from the pituitary, as well as its ability to bind activin.The asparagine and surrounding residues described in this analysis areweakly conserved between follistatin-1 and follistatin-3. There arehowever, two potential N-linked glycosylation sites in the sequence offollistatin-3 (N-73 and N-215; see FIG. 1A). In addition, 4 out of 5amino acids making up the sequence near the amino terminus, at whichpoint Inouye and coworkers made their two amino acid insertion (supra),are conserved. Consequently, the extreme amino terminal region of thepredicted mature follistatin-3 polypeptide may have a high potential forexhibiting a deleterious effect through mutation.

[0127] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of the follistatin-3 polypeptide can besubstantially purified by the one-step method described by Smith andJohnson (Gene 67:31-40 (1988)). Polypeptides of the invention also canbe purified from natural or recombinant sources using anti-Follistatin-3antibodies of the invention in methods which are well known in the artof protein purification.

[0128] The invention further provides an isolated follistatin-3polypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence of the full-lengthfollistatin-3 polypeptide having the complete amino acid sequence shownin SEQ ID NO:2 (i.e., positions −26 to 237 of SEQ ID NO:2); (b) theamino acid sequence of the full-length follistatin-3 polypeptide havingthe complete amino acid sequence shown in SEQ ID NO:2 excepting theN-terminal methionine (i.e., positions −25 to 237 of SEQ ID, NO:2); (c)the amino acid sequence of the predicted mature follistatin-3polypeptide having the amino acid sequence at positions 1 to 237 in SEQID NO:2; (d) the amino acid sequence of the full-length follistatin-3polypeptide having the complete amino acid sequence encoded by the cDNAclone contained in ATCC® Deposit No. 209199; (e) the amino acid sequenceof the full-length follistatin-3 polypeptide having the complete aminoacid sequence excepting the N-terminal methionine encoded by the cDNAclone contained in ATCC® Deposit No. 209199; and (f) the amino acidsequence of the mature follistatin-3 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC® Deposit No.209199. The polypeptides of the present invention also includepolypeptides having an amino acid sequence at least 80% identical, morepreferably at least 90% identical, and still more preferably 95%, 96%,97%, 98% or 99% identical to those described in (a), (b), (c), (d), (e)or (f) above, as well as polypeptides having an amino acid sequence withat least 90% similarity, and more preferably at least 95% similarity, tothose above.

[0129] Further polypeptides of the present invention includepolypeptides which have at least 90% similarity, more preferably atleast 95% similarity, and still more preferably at least 96%, 97%, 98%or 99% similarity to those described above. The polypeptides of theinvention also comprise those which are at least 80% identical, morepreferably at least 90% or 95% identical, still more preferably at least96%, 97%, 98% or 99% identical to the polypeptide encoded by thedeposited cDNA or to the polypeptide of SEQ ID NO:2, and also includeportions of such polypeptides with at least 30 amino acids and morepreferably at least 50 amino acids.

[0130] By “% similarity” for two polypeptides is intended a similarityscore produced by comparing the amino acid sequences of the twopolypeptides using the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711) and the defaultsettings for determining similarity. Bestfit uses the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics2:482-489, 1981) to find the best segment of similarity between twosequences.

[0131] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of afollistatin-3 polypeptide is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of the follistatin-3polypeptide. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

[0132] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence shown in FIG. 1A (SEQ ID NO:2), the amino acidsequence encoded by deposited cDNA clone HDTAH85, or fragments thereof,can be determined conventionally using known computer programs such theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

[0133] In a specific embodiment, the identity between a reference(query) sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, is determinedusing the FASTDB computer program based on the algorithm of Brutlag etal. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection is made to the results to take into consideration the factthat the FASTDB program does not account for N- and C-terminaltruncations of the subject sequence when calculating global percentidentity. For subject sequences truncated at the N- and C-termini,relative to the query sequence, the percent identity is corrected bycalculating the number of residues of the query sequence that are N- andC-terminal of the subject sequence, which are not matched/aligned with acorresponding subject residue, as a percent of the total bases of thequery sequence. A determination of whether a residue is matched/alignedis determined by results of the FASTDB sequence alignment. Thispercentage is then subtracted from the percent identity, calculated bythe above FASTDB program using the specified parameters, to arrive at afinal percent identity score. This final percent identity score is whatis-used for the purposes of this embodiment. Only residues to the N- andC-termini of the subject sequence, which are not matched/aligned withthe query sequence, are considered for the purposes of manuallyadjusting the percent identity score. That is, only query residuepositions outside the farthest N- and C-terminal residues of the subjectsequence. For example, a 90 amino acid residue subject sequence isaligned with a 100 residue query sequence to determine percent identity.The deletion occurs at the N-terminus of the subject sequence andtherefore, the FASTDB alignment does not show a matching/alignment ofthe first 10 residues at the N-terminus. The 10 unpaired residuesrepresent 10% of the sequence (number of residues at the N- andC-termini not matched/total number of residues in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 residues were perfectly matched thefinal percent identity would be 90%. In another example, a 90 residuesubject sequence is compared with a 100 residue query sequence. Thistime the deletions are internal deletions so there are no residues atthe N- or C-termini of the subject sequence which are notmatched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected for. No other manualcorrections are made for the purposes of this embodiment.

[0134] The invention also encompasses fusion proteins in which thefull-length follistatin-3 polypeptide or fragment, variant, derivative,or analog thereof is fused to an unrelated protein. These fusionproteins can be routinely designed on the basis of the follistatin-3nucleotide and polypeptide sequences disclosed herein. For example, asone of skill in the art will appreciate, follistatin-3 polypeptides andfragments (including epitope-bearing fragments) thereof described hereincan be combined with parts of the constant domain of immunoglobulins(IgG), resulting in chimeric (fusion) polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EP A 394,827; Traunecker, et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric follistatin-3polypeptide or polypeptide fragments alone (Fountoulakis, et al., J.Biochem. 270:3958-3964 (1995)). Examples of follistatin-3 fusionproteins that are encompassed by the invention include, but are notlimited to, fusion of the follistatin-3 polypeptide sequences to anyamino acid sequence that allows the fusion proteins to be displayed onthe cell surface (e.g. the IgG Fe domain); or fusions to an enzyme,fluorescent protein, or luminescent protein which provides a markerfunction.

[0135] The polypeptide of the present invention could be used as amolecular weight marker on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart.

[0136] As described in detail below, the polypeptides of the presentinvention can also be used to raise polyclonal and monoclonalantibodies, which are useful in assays for detecting follistatin-3protein expression as described below or as agonists and antagonistscapable of enhancing or inhibiting follistatin-3 protein function.Further, such polypeptides can be used in the yeast two-hybrid system to“capture” follistatin-3 protein binding proteins which are alsocandidate agonists and antagonists according to the present invention.The yeast two hybrid system is described by Fields and Song (Nature340:245-246 (1989)).

[0137] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of this polypeptide portion is an immunogenicor antigenic epitope of a polypeptide of the invention. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. On the other hand, aregion of a protein molecule to which an antibody can bind is defined asan “antigenic epitope”. The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes (see, forinstance, Geysen, et al., Proc. Natl. Acad. Sci. USA 81:3998-4002(1983)).

[0138] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein (see, for instance, Sutcliffe, J.G., et al., Science 219:660-666 (1983)). Peptides capable of elicitingprotein-reactive sera are frequently represented in the primary sequenceof a protein, can be characterized by a set of simple chemical rules,and are confined neither to immunodominant regions of intact proteins(i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention (see, forinstance, Wilson, et al., Cell 37:767-778 (1984)).

[0139] Antigenic epitope-bearing peptides and polypeptides of theinvention preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between about 15 to about30 amino acids contained within the amino acid sequence of a polypeptideof the invention. Non-limiting examples of antigenic polypeptides orpeptides that can be used to generate follistatin-3-specific antibodiesinclude: a polypeptide comprising amino acid residues from about Leu-14to about Ala-20, from about Ser-46 to about Ile-55, from about Gly-88 toabout Pro-97, from about Gly-113 to about Leu-133, from about Arg-138 toabout Glu-146, from about Pro-177 to about Thr-191, from about Gly-219to about Val-237. These polypeptide fragments have been determined tobear antigenic epitopes of the follistatin-3 protein by the analysis ofthe Jameson-Wolf antigenic index, as shown in FIG. 3 and Table I, above.

[0140] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means (see, for example, Houghten,R. A., et al., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985); and U.S.Pat. No. 4,631,211 to Houghten, et al. (1986)).

[0141] Epitope-bearing peptides and polypeptides of the invention areused to induce antibodies according to methods well known in the art(see, for instance, Sutcliffe, et al., supra; Wilson, et al., supra;Chow, M., et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F.J., et al., J. Gen. Virol. 66:2347-2354 (1985)). Immunogenicepitope-bearing peptides of the invention, i.e., those parts of aprotein that elicit an antibody response when the whole protein is theimmunogen, are identified according to methods known in the art (see,for instance, Geysen, et al., supra). Further still, U.S. Pat. No.5,194,392, issued to Geysen, describes a general method of detecting ordetermining the sequence of monomers (amino acids or other compounds)which is a topological equivalent of the epitope (i.e., a “mimotope”)which is complementary to a particular paratope (antigen binding site)of an antibody of interest. More generally, U.S. Pat. No. 4,433,092,issued to Geysen, describes a method of detecting or determining asequence of monomers which is a topographical equivalent of a ligandwhich is complementary to the ligand binding site of a particularreceptor of interest. Similarly, U.S. Pat. No. 5,480,971, issued toHoughten and colleagues, on Peralkylated Oligopeptide Mixtures discloseslinear C1-C7-alkyl peralkylated oligopeptides and sets and libraries ofsuch peptides, as well as methods for using such oligopeptide sets andlibraries for determining the sequence of a peralkylated oligopeptidethat preferentially binds to an acceptor molecule of interest. Thus,non-peptide analogs of the epitope-bearing peptides of the inventionalso can be made routinely by these methods.

[0142] As one of skill in the art will appreciate, follistatin-3polypeptides of the present invention and the epitope-bearing fragmentsthereof described above can be combined with parts of the constantdomain of immunoglobulins (IgG), resulting in chimeric polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (EP A 394,827; Traunecker, et al., Nature331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric follistatin-3 protein orprotein fragment alone (Fountoulakis, et al., J. Biochem. 270:3958-3964(1995)).

[0143] Follistatin-3 protein-specific antibodies for use in the presentinvention can be raised against the intact follistatin-3 protein or anantigenic polypeptide fragment thereof, which may be presented togetherwith a carrier protein, such as an albumin, to an animal system (such asrabbit or mouse) or, if it is long enough (at least about 25 aminoacids), without a carrier.

[0144] Antibodies

[0145] Further polypeptides of the invention relate to antibodies andT-cell antigen receptors (TCR) which immunospecifically bind apolypeptide, polypeptide fragment, or variant of SEQ ID NO:2, and/or anepitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0146] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

[0147] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

[0148] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

[0149] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homolog of a polypeptide of thepresent invention are included. Antibodies that bind polypeptides withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. In specific embodiments, antibodies of thepresent invention cross-react with murine, rat and/or rabbit homologs ofhuman proteins and the corresponding epitopes thereof. Antibodies thatdo not bind polypeptides with less than 95%, less than 90%, less than85%, less than 80%, less than 75%, less than 70%, less than 65%, lessthan 60%, less than 55%, and less than 50% identity (as calculated usingmethods known in the art and described herein) to a polypeptide of thepresent invention are also included in the present invention. In aspecific embodiment, the above-described cross-reactivity is withrespect to any single specific antigenic or immunogenic polypeptide, orcombination(s) of 2, 3, 4, 5, or more of the specific antigenic and/orimmunogenic polypeptides disclosed herein. Further included in thepresent invention are antibodies which bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁷M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹² M, ¹⁰⁻¹² M, 5×10⁻¹³ M, 10¹³M, 5×10¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵M, or 10⁻¹⁵ M.

[0150] The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

[0151] Antibodies of the present invention may act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Preferrably, antibodies of the presentinvention bind an antigenic epitope disclosed herein, or a portionthereof. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody.

[0152] The invention also features receptor-specific antibodies whichboth prevent ligand binding and receptor activation as well asantibodies that recognize the receptor-ligand complex, and, preferably,do not specifically recognize the unbound receptor or the unboundligand. Likewise, included in the invention are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

[0153] Antibodies of the present invention may be used, for example, butnot limited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

[0154] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

[0155] The antibodies of the invention include derivatives that aremodified, i.e, by the covalent attachment of any type of molecule to theantibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

[0156] The antibodies of the present invention may be generated by anysuitable method known in the art. Polyclonal antibodies to anantigen-of-interest can be produced by various procedures well known inthe art. For example, a polypeptide of the invention can be administeredto various host animals including, but not limited to, rabbits, mice,rats, etc. to induce the production of sera containing poyclonalantibodies specific for the antigen. Various adjuvants may be used toincrease the immunological response, depending on the host species, andinclude but are not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

[0157] Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

[0158] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples (e.g., Example 20). In anon-limiting example, mice can be immunized with a polypeptide of theinvention or a cell expressing such peptide. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC®. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

[0159] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0160] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CHI domain ofthe heavy chain.

[0161] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

[0162] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

[0163] Examples of techniques which can be used to produce single-chainFvs and antibodies include those described in U.S. Pat. Nos. 4,946,778and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991);Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science240:1038-1040 (1988). For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. A chimeric antibody is amolecule in which different portions of the antibody are derived fromdifferent animal species, such as antibodies having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, whichare incorporated herein by reference in their entirety. Humanizedantibodies are antibody molecules from non-human species antibody thatbinds the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and a framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

[0164] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety.

[0165] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

[0166] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

[0167] Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

[0168] Polynucleotides Encoding Antibodies

[0169] The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or lower stringency hybridization conditions, e.g., asdefined supra, to polynucleotides that encode an antibody, preferably,that specifically binds to a polypeptide of the invention, preferably,an antibody that binds to a polypeptide having the amino acid sequenceof SEQ ID NO:2.

[0170] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0171] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be chemically synthesized orobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

[0172] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

[0173] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well know in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

[0174] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

[0175] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42(1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);and Ward et al., Nature 334:544-54 (1989)) can be adapted to producesingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

[0176] Methods of Producing Antibodies

[0177] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

[0178] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

[0179] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof, or asingle chain antibody of the invention, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

[0180] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression-vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter clement from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

[0181] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0182] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0183] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

[0184] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

[0185] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0186] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

[0187] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol.3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

[0188] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52(1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

[0189] Once an antibody molecule of the invention has been produced byan animal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

[0190] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

[0191] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CHI domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fe portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fe portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

[0192] As discussed, supra, the polypeptides corresponding to apolypeptide, polypeptide fragment, or a variant of SEQ ID NO:2 may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides corresponding to SEQID NO:2 may be fused or conjugated to the above antibody portions tofacilitate purification.

[0193] One reported example describes chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86(1988). The polypeptides of the present invention fused or conjugated toan antibody having disulfide-linked dimeric structures (due to the IgG)may also be more efficient in binding and neutralizing other molecules,than the monomeric secreted protein or protein fragment alone.(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases,the Fe part in a fusion protein is beneficial in therapy and diagnosis,and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fe part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fe portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fe portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

[0194] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell37:767 (1984)) and the “flag” tag.

[0195] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

[0196] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²³Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0197] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0198] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

[0199] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

[0200] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0201] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

[0202] Immunophenotyping

[0203] The antibodies of the invention may be utilized forimmunophenotyping of cell lines and biological samples. The translationproduct of the gene of the present invention may be useful as a cellspecific marker, or more specifically as a cellular marker that isdifferentially expressed at various stages of differentiation and/ormaturation of particular cell types. Monoclonal antibodies directedagainst a specific epitope, or combination of epitopes, will allow forthe screening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0204] These techniques allow for the screening of particularpopulations of cells, such as might be found with hematologicalmalignancies (i.e. minimal residual disease (MRD) in acute leukemicpatients) and “non-self” cells in transplantations to preventGraft-versus-Host Disease (GVHD). Alternatively, these techniques allowfor the screening of hematopoietic stem and progenitor cells capable ofundergoing proliferation and/or differentiation, as might be found inhuman umbilical cord blood.

[0205] Assays for Antibody Binding

[0206] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0207] immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C, washing the beads in lysisbuffer and resuspending the beads in SDS/sample buffer. The ability ofthe antibody of interest to immunoprecipitate a particular antigen canbe assessed by, e.g., western blot analysis. One of skill in the artwould be knowledgeable as to the parameters that can be modified toincrease the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0208] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., 32P or 1251) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0209] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0210] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., 3H or 125I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest conjugated to alabeled compound (e.g., ³H or ¹²⁵I) in the presence of increasingamounts of an unlabeled second antibody.

[0211] Therapeutic Uses

[0212] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the disclosed diseases, disorders, orconditions. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0213] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0214] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0215] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

[0216] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10-2M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

[0217] Gene Therapy

[0218] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

[0219] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0220] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596(1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0221] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

[0222] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0223] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

[0224] In a specific embodiment, viral vectors that contains nucleicacid sequences encoding an antibody of the invention are used. Forexample, a retroviral vector can be used (see Miller et al., Meth.Enzymol. 217:581-599 (1993)). These retroviral vectors contain thecomponents necessary for the correct packaging of the viral genome andintegration into the host cell DNA. The nucleic acid sequences encodingthe antibody to be used in gene therapy are cloned into one or morevectors, which facilitates delivery of the gene into a patient. Moredetail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993).

[0225] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

[0226] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0227] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0228] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0229] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0230] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0231] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0232] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0233] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. Demonstration of Therapeutic or ProphylacticActivity

[0234] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0235] Therapeutic/Prophylactic Administration and Composition

[0236] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably anantibody of the invention. In a preferred aspect, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

[0237] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0238] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0239] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0240] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0241] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

[0242] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0243] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0244] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0245] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0246] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0247] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0248] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0249] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0250] Diagnosis and Imaging

[0251] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

[0252] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0253] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (⁹⁹Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0254] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0255] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99 mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0256] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0257] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0258] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRJ), and sonography.

[0259] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

[0260] Kits

[0261] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0262] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0263] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0264] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0265] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigma, St. Louis, Mo.).

[0266] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0267] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

[0268] Reproductive System- and Cell Growth and Differentiation-RelatedDisorders Diagnosis

[0269] The present inventors have discovered that follistatin-3 isexpressed not only in Hodgkin's Lymphoma, but also in synovialfibroblasts, gall bladder, resting and serum-induced smooth muscle,testes, Merkel cells, HEL cells, hippocampus, TNF-alpha- and IFN-inducedepithelial cells, keratinocyte, amygdala depression, HL-60 cells,hepatoma, progesterone-treated epidermal cells, endothelial cells,HSC172 cells, epithelioid sarcoma, activated T-cells, breast lymph node,pancreatic carcinoma, fetal dura mater, fetal lung, epididymis,placenta, dendritic cells, rejected kidney, and uterine cancer. For anumber of reproductive system-related disorders and disorders related tothe regulation of cell growth and differentiation, substantially altered(increased or decreased) levels of follistatin-3 gene expression can bedetected in reproductive system tissue or other cells or bodily fluids(e.g., sera, plasma, urine, synovial fluid or spinal fluid) taken froman individual having such a disorder, relative to a “standard”follistatin-3 gene expression level, that is, the follistatin-3expression level in reproductive system tissues or bodily fluids from anindividual not having the reproductive system or cell growth anddifferentiation disorder. Thus, the invention provides a diagnosticmethod useful during diagnosis of a reproductive system or cell growthand differentiation disorder, which involves measuring the expressionlevel of the gene encoding the follistatin-3 protein in reproductivesystem tissue or other cells or body fluid from an individual andcomparing the measured gene expression level with a standardfollistatin-3 gene expression level, whereby an increase or decrease inthe gene expression level compared to the standard is indicative of areproductive or cell growth and differentiation system disorder.

[0270] In particular, it is believed that certain tissues in mammalswith cancer of various cells and tissues of the reproductive or othersystems express significantly reduced levels of the follistatin-3protein and mRNA encoding the follistatin-3 protein when compared to acorresponding “standard” level. Further, it is believed that enhancedlevels of the follistatin-3 protein can be detected in certain bodyfluids (e.g., sera, plasma, urine, and spinal fluid) from mammals withsuch a cancer when compared to sera from mammals of the same species nothaving the cancer.

[0271] Thus, the invention provides a diagnostic method useful duringdiagnosis of reproductive system or cell growth and differentiationdisorders, including cancers of these systems, which involves measuringthe expression level of the gene encoding the follistatin-3 protein inreproductive system tissue or other cells or body fluid from anindividual and comparing the measured gene expression level with astandard follistatin-3 gene expression level, whereby an increase ordecrease in the gene expression level compared to the standard isindicative of a reproductive system disorder or a disorder of theregulation of cell growth and differentiation.

[0272] Where a diagnosis of a disorder in the reproductive or othersystem including diagnosis of a tumor, has already been made accordingto conventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting depressed follistatin-3 geneexpression will experience a worse clinical outcome relative to patientsexpressing the gene at a level nearer the standard level.

[0273] By “assaying the expression level of the gene encoding thefollistatin-3 protein” is intended qualitatively or quantitativelymeasuring or estimating the level of the follistatin-3 protein or thelevel of the mRNA encoding the follistatin-3 protein in a firstbiological sample either directly (e.g., by determining or estimatingabsolute protein level or mRNA level) or relatively (e.g., by comparingto the follistatin-3 protein level or mRNA level in a second biologicalsample). Preferably, the follistatin-3 protein level or mRNA level inthe first biological sample is measured or estimated and compared to astandard follistatin-3 protein level or mRNA level, the standard beingtaken from a second biological sample obtained from an individual nothaving the disorder or being determined by averaging levels from apopulation of individuals not having a disorder of the reproductivesystem or of regulation of cell growth and differentiation. As will beappreciated in the art, once a standard follistatin-3 protein level ormRNA level is known, it can be used repeatedly as a standard forcomparison.

[0274] By “biological sample” is intended any biological sample obtainedfrom an individual, body fluid, cell line, tissue culture, or othersource which contains follistatin-3 protein or mRNA. As indicated,biological samples include body fluids (such as sera, plasma, urine,synovial fluid and spinal fluid) which contain free follistatin-3protein, reproductive system tissue, and other tissue sources found toexpress complete or mature follistatin-3 or a follistatin-3 receptor.Methods for obtaining tissue biopsies and body fluids from mammals arewell known in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

[0275] The present invention is useful for diagnosis or treatment ofvarious reproductive system-related disorders and disorders of theregulation of cell growth and differentiation in mammals, preferablyhumans. Such disorders include tumors, cancers, interstitial lungdisease, and any disregulation of the growth and differentiationpatterns of cell function including, but not limited to, autoimmunity,arthritis, leukemias, lymphomas, immunosuppression, immunity, humoralimmunity, inflammatory bowel disease, myelosuppression and the like.

[0276] Total cellular RNA can be isolated from a biological sample usingany suitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described byChomczynski and Sacchi (Anal. Biochem. 162:156-159 (1987)). Levels ofmRNA encoding the follistatin-3 protein are then assayed using anyappropriate method. These include Northern blot analysis, S1 nucleasemapping, the polymerase chain reaction (PCR), reverse transcription incombination with the polymerase chain reaction (RT-PCR), and reversetranscription in combination with the ligase chain reaction (RT-LCR).

[0277] Assaying follistatin-3 protein levels in a biological sample canoccur using antibody-based techniques. For example, follistatin-3protein expression in tissues can be studied with classicalimmunohistological methods (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting follistatin-3protein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase, and radioisotopes, such as iodine (¹²⁵I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), andtechnetium (^(99m)Tc), and fluorescent labels, such as fluorescein andrhodamine, and biotin.

[0278] In addition to assaying follistatin-3 protein levels in abiological sample obtained from an individual, follistatin-3 protein canalso be detected in vivo by imaging. Antibody labels or markers for invivo imaging of follistatin-3 protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

[0279] A follistatin-3 protein-specific antibody or antibody fragmentwhich has been labeled with an appropriate detectable imaging moiety,such as a radioisotope (for example, ¹³¹I, ¹¹² In, ^(99m)Tc), aradio-opaque substance, or a material detectable by nuclear magneticresonance, is introduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain follistatin-3 protein.In vivo tumor imaging is described by Burchiel and coworkers (Chapter 13in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel, S. W.and Rhodes, B. A., eds., Masson Publishing Inc. (1982)). Treatment

[0280] As noted above, follistatin-3 polynucleotides and polypeptidesare useful for diagnosis of conditions involving abnormally high or lowexpression of follistatin-3 activities. Given the cells and tissueswhere follistatin-3 is expressed as well as the activities modulated byfollistatin-3, it is readily apparent that a substantially altered(increased or decreased) level of expression of follistatin-3 in anindividual compared to the standard or “normal” level producespathological conditions related to the bodily system(s) in whichfollistatin-3 is expressed and/or is active.

[0281] It will also be appreciated by one of ordinary skill that, sincethe follistatin-3 protein of the invention is a member of theinhibin-related protein family the mature secreted form of the proteinmay be released in soluble form from the cells which express thefollistatin-3 by proteolytic cleavage. Therefore, when follistatin-3mature form is added from an exogenous source to cells, tissues or thebody of an individual, the protein will exert its physiologicalactivities on its target cells of that individual.

[0282] Therefore, it will be appreciated that conditions caused by adecrease in the standard or normal level of Follistatin-3 activity in anindividual, particularly disorders of the reproductive system, can betreated by administration of follistatin-3 polypeptide (in the form ofthe mature protein). Thus, the invention also provides a method oftreatment of an individual in need of an increased level offollistatin-3 activity comprising administering to such an individual apharmaceutical composition comprising an amount of an isolatedfollistatin-3 polypeptide of the invention, particularly a mature formof the follistatin-3 protein of the invention, effective to increase thefollistatin-3 activity level in such an individual.

[0283] Follistatin-3 may be used to treat male sterility by its innateability to bind activin and, as a result, prevent activin-binding to itsreceptor. Activin receptor-binding results in a suppression of FSHsecretion. Increased levels of FSH, in turn, result in an increase inspermatogenesis (Ying, S.-Y. Enclocrine Rev. 9:267-293 (1988)). Thus, adecrease in the effective concentration of activin will result in anFSH-mediated increase in spermatogenesis. In addition, since activinelicits a number of biological effects including the modulation ofgonadal androgen biosynthesis (Hsueh, A. J. W., et al., Proc. Natl.Acad. Sci. USA 84:5082-5086 (1987)), the attenuation of growth hormonesecretion (Bilezikjian, L. M., et al., Endocrinology 126:2369-2376(1990), the promotion of erythroid cell differentiation (Eto, Y., etal., Biochem. Biophys. Res. Comm. 142:1095-1103 (1987)), the inductionof mesoderm formation (Smith, J. C., et al., Nature 345:729-731 (1990)),and the maintenance of nerve cell survival (Schubert, D., et al., Nature344:868-870 (1990)), and since follistatin-3 directly inhibits activinacitivity, follistatin-3 may be used to therapeutically regulate, aswell as diagnostically evaluate, the conditions and events listed above.Follistatin-3 may also be used to inhibit the activin-induceddifferentiation of follicular granulosa cells (Nakamura, T., et al.,Biochim. Biophys. Acta 1135:103-109 (1992)). Follistatin-3 may be usedtherapeutically to regulate autocrine endothelial cell activity and, asa result, induce angiogenesis (Kozian, D. H., et al., Lab. Invest.76:267-276 (1997)). Follistatin-3 may also be used to inhibit theactivity of activin and thereby prevent the observed activin-mediatedinhibition of basal and androgen-stimulated proliferation and inductionof apoptosis (Wang, Q. F., et al., Endocrinology 137:5476-5483 (1996)).Treatment to increase the expression or the presence of follistatin-3protein may be used to inhibit the progression of gonadotroph adenomas,osteosarcomas, hepatomas, and other tumors and cancers including bone,breast, colon, lymphomas, leukemias, epithelial carcinomas, pancreatic,stomach, liver, lung, melanoma, prostate, ovarian, uterine, bladder,gliomas, retinoblastomas, sarcomas, and the like (Penabad, J. L., etal., J. Clin. Endocrinol. Metab. 81:3397-3403 (1996); Kato, M. V., etal., Oncogene 12:1361-1364 (1996)). Follistatin-3 may also be employedto stimulate wound healing. In this same manner, follistatin-3 may alsobe employed to treat other fibrotic disorders, including livercirrhosis, osteoarthritis and pulmonary fibrosis. Follistatin-3 alsoincreases the presence of eosinophils which have the distinctivefunction of killing the larvae of parasites that invade tissues, as inschistosomiasis, trichinosis and ascariasis. It may also be employed toregulate hematopoiesis, by regulating the activation and differentiationof various hematopoietic progenitor cells, for example, to releasemature leukocytes from the bone marrow following chemotherapy, i.e., instem cell mobilization. Follistatin-3 may also be employed to treatsepsis. Follistatin-3 may also be used to treat a number of diseasestates known to those of skill in the art which may be therapeuticallyregulated by exploiting the prohibitive interation of follistatin-3 withthe activin molecule.

[0284] In certain embodiments, follistatin-3 and/or agonists orantagonists of follistatin-3 may be used to treat cancers including, butnot limited to those described herein. In one embodiment, the cancer isB cell chronic lymphocytic leukemia. In a further embodiment, the canceris a non-Hodgkin lymphoma.

[0285] Formulations

[0286] The follistatin-3 polypeptide composition will be formulated anddosed in a fashion consistent with good medical practice, taking intoaccount the clinical condition of the individual patient (especially theside effects of treatment with follistatin-3 polypeptide alone), thesite of delivery of the follistatin-3 polypeptide composition, themethod of administration, the scheduling of administration, and otherfactors known to practitioners. The “effective amount” of follistatin-3polypeptide for purposes herein is thus determined by suchconsiderations.

[0287] As a general proposition, the total pharmaceutically effectiveamount of follistatin-3 polypeptide administered parenterally per dosewill be in the range of about 1 μg/kg/day to 10 mg/kg/day of patientbody weight, although, as noted above, this will be subject totherapeutic discretion. More preferably, this dose is at least 0.01mg/kg/day, and most preferably for humans between about 0.01 and 1mg/kg/day for the hormone. If given continuously, the follistatin-3polypeptide is typically administered at a dose rate of about 1μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day orby continuous subcutaneous infusions, for example, using a mini-pump. Anintravenous bag solution may also be employed. The length of treatmentneeded to observe changes and the interval following treatment forresponses to occur appears to vary depending on the desired effect.

[0288] Pharmaceutical compositions containing the follistatin-3 of theinvention may be administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), bucally, or as an oralor nasal spray. By “pharmaceutically acceptable carrier” is meant anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[0289] The follistatin-3 polypeptide is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U.,et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethylmethacrylate; Langer, R., et al., J. Biomed. Mater. Res. 15:167-277(1981), and Langer, R., Chem. Tech. 12:98-105 (1982)), ethylene vinylacetate (Langer, R., et al., Id.) or poly-D-(−)-3-hydroxybutyric acid(EP 133,988). Sustained-release follistatin-3 polypeptide compositionsalso include liposomally entrapped follistatin-3 polypeptide. Liposomescontaining follistatin-3 polypeptide are prepared by methods known inthe art (DE 3,218,121; Epstein, et al., Proc. Natl. Acad. Sci. (USA)82:3688-3692 (1985); Hwang, et al., Proc. Natl. Acad. Sci. (USA)77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324). Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal follistatin-3 polypeptide therapy.

[0290] For parenteral administration, in one embodiment, thefollistatin-3 polypeptide is formulated generally by mixing it at thedesired degree of purity, in a unit dosage injectable form (solution,suspension, or emulsion), with a pharmaceutically acceptable carrier,i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the formulation preferably does not includeoxidizing agents and other compounds that are known to be deleterious topolypeptides.

[0291] Generally, the formulations are prepared by contacting thefollistatin-3 polypeptide uniformly and intimately with liquid carriersor finely divided solid carriers or both. Then, if necessary, theproduct is shaped into the desired formulation. Preferably the carrieris a parenteral carrier, more preferably a solution that is isotonicwith the blood of the recipient. Examples of such carrier vehiclesinclude water, saline, Ringer's solution, and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate are also usefulherein, as well as liposomes.

[0292] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0293] The follistatin-3 polypeptide is typically formulated in suchvehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the useof certain of the foregoing excipients, carriers, or stabilizers willresult in the formation of follistatin-3 polypeptide salts.

[0294] Follistatin-3 polypeptide to be used for therapeuticadministration must be sterile. Sterility is readily accomplished byfiltration through sterile filtration membranes (e.g., 0.2 micronmembranes). Therapeutic follistatin-3 polypeptide compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

[0295] Follistatin-3 polypeptide ordinarily will be stored in unit ormulti-dose containers, for example, sealed ampoules or vials, as anaqueous solution or as a lyophilized formulation for reconstitution. Asan example of a lyophilized formulation, 10-ml vials are filled with 5ml of sterile-filtered 1% (w/v) aqueous follistatin-3 polypeptidesolution, and the resulting mixture is lyophilized. The infusionsolution is prepared by reconstituting the lyophilized follistatin-3polypeptide using bacteriostatic water-for-injection (WFI).

[0296] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides of the present invention may be employed inconjunction with other therapeutic compounds.

[0297] Agonists and Antagonists—Assays and Molecules

[0298] The invention also provides a method of screening compounds toidentify those which enhance or block the action of follistatin-3 oncells, such as its interaction with follistatin-3-binding molecules suchas activin, an activin-like molecule, or a follistatin-3 receptormolecule. An agonist is a compound which increases the naturalbiological functions of follistatin-3 or which functions in a mannersimilar to follistatin-3, while antagonists decrease or eliminate suchfunctions.

[0299] In another aspect of this embodiment the invention provides amethod for identifying an activin-like-molecule or a receptor protein orother ligand-binding protein which binds specifically to a follistatin-3polypeptide. For example, a cellular compartment, such as a membrane ora preparation thereof, may be prepared from a cell that expresses amolecule that binds follistatin-3. The preparation is incubated withlabeled follistatin-3 and complexes of follistatin-3 bound to theactivin-like molecule, receptor or other binding protein are isolatedand characterized according to routine methods known in the art.Alternatively, the follistatin-3 polypeptide may be bound to a solidsupport so that binding molecules solubilized from cells are bound tothe column and then eluted and characterized according to routinemethods.

[0300] In the assay of the invention for agonists or antagonists, acellular compartment, such as a membrane or a preparation thereof, maybe prepared from a cell that expresses a molecule that bindsfollistatin-3, such as a molecule of a signaling or regulatory pathwaymodulated by follistatin-3. The preparation is incubated with labeledfollistatin-3 in the absence or the presence of a candidate moleculewhich may be a follistatin-3 agonist or antagonist. The ability of thecandidate molecule to bind the binding molecule is reflected indecreased binding of the labeled ligand. Molecules which bindgratuitously, i.e., without inducing the effects of follistatin-3 onbinding the follistatin-3 binding molecule, are most likely to be goodantagonists. Molecules that bind well and elicit effects that are thesame as or closely related to follistatin-3 are agonists.

[0301] Follistatin-3-like effects of potential agonists and antagonistsmay by measured, for instance, by determining activity of a secondmessenger system following interaction of the candidate molecule with acell or appropriate cell preparation, and comparing the effect with thatof follistatin-3 or molecules that elicit the same effects asfollistatin-3. Second messenger systems that may be useful in thisregard include but are not limited to AMP guanylate cyclase, ion channelor phosphoinositide hydrolysis second messenger systems.

[0302] Another example of an assay for follistatin-3 antagonists is acompetitive assay that combines follistatin-3 and a potential antagonistwith membrane-bound follistatin-3 receptor molecules or recombinantfollistatin-3 receptor molecules under appropriate conditions for acompetitive inhibition assay. Follistatin-3 can be labeled, such as byradioactivity, such that the number of follistatin-3 molecules bound toa receptor molecule can be determined accurately to assess theeffectiveness of the potential antagonist.

[0303] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducingfollistatin-3-induced activities, thereby preventing the action offollistatin-3 by excluding follistatin-3 from binding.

[0304] Other potential antagonists include antisense molecules.Antisense technology can be used to control gene expression throughantisense DNA or RNA or through triple-helix formation. Antisensetechniques are discussed in a number of studies (for example, Okano, J.Neurochem. 56:560 (1991); “Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression.” CRC Press, Boca Raton, Fla. (1988)). Triple helixformation is discussed in a number of studies, as well (for instance,Lee, et al., Nucleic Acids Research 6:3073 (1979); Cooney, et al.,Science 241:456 (1988); Dervan, et al., Science 251:1360 (1991)). Themethods are based on binding of a polynucleotide to a complementary DNAor RNA. For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of follistatin-3. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into follistatin-3 polypeptide. The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production offollistatin-3 protein.

[0305] The agonists and antagonists may be employed in a compositionwith a pharmaceutically acceptable carrier, e.g., as described above.

[0306] Antagonists of follistatin-3 may be employed, for instance, totreat a deficiency in FSH, estrogen, and other hormones. Follistatin-1and follistatin-3 are potent inhibitors of FSH and estrogen productionand secretion. As a result, a deficiency of these or related hormonesmay be corrected or ameliorated through the use of a follistatin-3antagonist. A follistatin-3 antagonist may be used to prevent or inhibitor reduce the production of spermatozoa by inhibiting the interaction offollistatin-3 with activin. Antagonists of follistatin-3 may also beused to modulate gonadal androgen biosynthesis, attenuate growth hormonesecretion, promote the differentiation of follicular granulosa,erythroid, and other cell types, induce mesoderm formation, and increasethe survival of nerve cells. A follistatin-3 antagonist may be used toinhibit angiogenesis related to or independent of tumorigenesis.Follistatin-3 antagonists may also be useful in increasing the activityof activin and thereby increasing the observed activin-mediatedinhibition of basal and androgen-stimulated proliferation and inductionof apoptosis. Antagonists of follistatin-3 may be used to regulate thehormonal and growth factor environment, and consequently, the activityof macrophages and their precursors, and of neutrophils, basophils, Blymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic Tcells and natural killer cells, in certain auto-immune and chronicinflammatory and infective diseases. Examples of auto-immune diseasesinclude multiple sclerosis, and insulin-dependent diabetes. Theantagonists may also be employed to treat infectious diseases includingsilicosis, sarcoidosis, idiopathic pulmonary fibrosis by alterring theactivation state of mononuclear phagocytes. They may also be employed totreat idiopathic hyper-eosinophilic syndrome by preventing eosinophilproduction and activation. Endotoxic shock may also be treated by theantagonists by preventing the activation of macrophages. Any of theabove antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

[0307] Gene Mapping

[0308] The nucleic acid molecules of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. Moreover, there is a current need foridentifying particular sites on the chromosome. Few chromosome markingreagents based on actual sequence data (repeat polymorphisms) arepresently available for marking chromosomal location. The mapping ofDNAs to chromosomes according to the present invention is an importantfirst step in correlating those sequences with genes associated withdisease.

[0309] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a follistatin-3 protein gene.This can be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose.

[0310] In addition, in some cases, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3′ untranslated region of the gene isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Fluorescence in situhybridization (“FISH”) of a cDNA clone to a metaphase chromosomal spreadcan be used to provide a precise chromosomal location in one step. Thistechnique can be used with probes from the cDNA as short as 50 or 60 bp(for a review of this technique, see Verma, et al., Human Chromosomes: AManual Of Basic Techniques, Pergamon Press, New York (1988)).

[0311] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, onthe World Wide Web (McKusick, V. Mendelian Inheritance In Man, availableon-line through Johns Hopkins University, Welch Medical Library). Therelationship between genes and diseases that have been mapped to thesame chromosomal region are then identified through linkage analysis(coinheritance of physically adjacent genes).

[0312] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0313] Exemplary Uses of the Follistatin-3 Polynucleotides.

[0314] The follistatin-3 polynucleotides identified herein can be usedin numerous ways as reagents. The following description should beconsidered exemplary and utilizes known techniques.

[0315] There exists an ongoing need to identify new chromosome markers,since few chromosome marking reagents, based on actual sequence data(repeat polymorphisms), are presently available.

[0316] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:1.Primers can be selected using computer analysis so that primers do notspan more than one predicted exon in the genomic DNA. These primers arethen used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humanfollistatin-3 gene corresponding to the SEQ ID NO:1 will yield anamplified fragment.

[0317] Similarly, somatic hybrids provide a rapid method of PCR mappingthe polynucleotides to particular chromosomes. Three or more clones canbe assigned per day using a single thermal cycler. Moreover,sublocalization of the follistatin-3 polynucleotides can be achievedwith panels of specific chromosome fragments. Other gene mappingstrategies that can be used include in situ hybridization, prescreeningwith labeled flow-sorted chromosomes, and preselection by hybridizationto construct chromosome specific-cDNA libraries.

[0318] Precise chromosomal location of the follistatin-3 polynucleotidescan also be achieved using fluorescence in situ hybridization (FISH) ofa metaphase chromosomal spread. This technique uses polynucleotides asshort as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp arepreferred. For a review of this technique, see Verma et al., “HumanChromosomes: a Manual of Basic Techniques,” Pergamon Press, New York(1988).

[0319] For chromosome mapping, the follistatin-3 polynucleotides can beused individually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes). Preferred polynucleotides correspond to the noncodingregions of the cDNAs because the coding sequences are more likelyconserved within gene families, thus increasing the chance of crosshybridization during chromosomal mapping.

[0320] Once a polynucleotide has been mapped to a precise chromosomallocation, the physical position of the polynucleotide can be used inlinkage analysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

[0321] Thus, once coinheritance is established, differences in thefollistatin-3 polynucleotide and the corresponding gene between affectedand unaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the follistatin-3 polypeptide and thecorresponding gene from several normal individuals is required todistinguish the mutation from a polymorphism. If a new polymorphism isidentified, this polymorphic polypeptide can be used for further linkageanalysis.

[0322] Furthermore, increased or decreased expression of the gene inaffected individuals as compared to unaffected individuals can beassessed using follistatin-3 polynucleotides. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

[0323] In addition to the foregoing, a follistatin-3 polynucleotide canbe used to control gene expression through triple helix formation orantisense DNA or RNA. Both methods rely on binding of the polynucleotideto DNA or RNA. For these techniques, preferred polynucleotides areusually 20 to 40 bases in length and complementary to either the regionof the gene involved in transcription (triple helix—see Lee et al.,Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988);and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself(antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988).) Triple helix formation optimally results in a shut-off of RNAtranscription from DNA, while antisense RNA hybridization blockstranslation of an mRNA molecule into polypeptide. Both techniques areeffective in model systems, and the information disclosed herein can beused to design antisense or triple helix polynucleotides in an effort totreat disease.

[0324] Follistatin-3 polynucleotides are also useful in gene therapy.One goal of gene therapy is to insert a normal gene into an organismhaving a defective gene, in an effort to correct the genetic defect.Follistatin-3 offers a means of targeting such genetic defects in ahighly accurate manner. Another goal is to insert a new gene that wasnot present in the host genome, thereby producing a new trait in thehost cell.

[0325] The follistatin-3 polynucleotides are also useful for identifyingindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The follistatin-3 polynucleotides canbe used as additional DNA markers for RFLP.

[0326] The follistatin-3 polynucleotides can also be used as analternative to RFLP, by determining the actual base-by-base DNA sequenceof selected portions of an individual's genome. These sequences can beused to prepare PCR primers for amplifying and isolating such selectedDNA, which can then be sequenced. Using this technique, individuals canbe identified because each individual will have a unique set of DNAsequences. Once an unique ID database is established for an individual,positive identification of that individual, living or dead, can be madefrom extremely small tissue samples.

[0327] Forensic biology also benefits from using DNA-basedidentification techniques as disclosed herein. DNA sequences taken fromvery small biological samples such as tissues, e.g., hair or skin, orbody fluids, e.g., blood, saliva, semen, etc., can be amplified usingPCR. In one prior art technique, gene sequences amplified frompolymorphic loci, such as DQa class II HLA gene, are used in forensicbiology to identify individuals. (Erlich, H., PCR Technology, Freemanand Co. (1992).) Once these specific polymorphic loci are amplified,they are digested with one or more restriction enzymes, yielding anidentifying set of bands on a Southern blot probed with DNAcorresponding to the DQa class II HLA gene. Similarly, follistatin-3polynucleotides can be used as polymorphic markers for forensicpurposes.

[0328] There is also a need for reagents capable of identifying thesource of a particular tissue. Such need arises, for example, inforensics when presented with tissue of unknown origin. Appropriatereagents can comprise, for example, DNA probes or primers specific toparticular tissue prepared from follistatin-3 sequences. Panels of suchreagents can identify tissue by species and/or by organ type. In asimilar fashion, these reagents can be used to screen tissue culturesfor contamination.

[0329] Because follistatin-3 is found expressed in Hodgkin's lymphoma,and a variety of additional cells and tissue types detailed infra,follistatin-3 polynucleotides are useful as hybridization probes fordifferential identification of the tissue(s) or cell type(s) present ina biological sample. Similarly, polypeptides and antibodies directed tofollistatin-3 polypeptides are useful to provide immunological probesfor differential identification of the tissue(s) or cell type(s). Inaddition, for a number of disorders of the above tissues or cells,particularly of the reproductive systems, significantly higher or lowerlevels of follistatin-3 gene expression may be detected in certaintissues (e.g., cancerous and wounded tissues) or bodily fluids (e.g.,serum, plasma, urine, synovial fluid or spinal fluid) taken from anindividual having such a disorder, relative to a “standard”follistatin-3 gene expression level, i.e., the follistatin-3 expressionlevel in healthy tissue from an individual not having the reproductivesystem disorder.

[0330] Thus, the invention provides a diagnostic method of a disorder,which involves: (a) assaying follistatin-3 gene expression level incells or body fluid of an individual; (b) comparing the follistatin-3gene expression level with a standard follistatin-3 gene expressionlevel, whereby an increase or decrease in the assayed follistatin-3 geneexpression level compared to the standard expression level is indicativeof disorder in the reproductive system.

[0331] In the very least, the follistatin-3 polynucleotides can be usedas molecular weight markers on Southern gels, as diagnostic probes forthe presence of a specific mRNA in a particular cell type, as a probe to“subtract-out” known sequences in the process of discovering novelpolynucleotides, for selecting and making oligomers for attachment to a“gene chip” or other support, to raise anti-DNA antibodies using DNAimmunization techniques, and as an antigen to elicit an immune response.

[0332] Exemplary Uses of Follistatin-3 Polypeptides.

[0333] Follistatin-3 polypeptides can be used in numerous ways. Thefollowing description should be considered exemplary and utilizes knowntechniques.

[0334] Follistatin-3 polypeptides can be used to assay protein levels ina biological sample using antibody-based techniques. For example,protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (1251, 1211), carbon (14C),sulfur (35S), tritium (3H), indium (¹¹²In), and technetium (99 mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

[0335] In addition to assaying secreted protein levels in a biologicalsample, proteins can also be detected in vivo by imaging. Antibodylabels or markers for in vivo imaging of protein include thosedetectable by X-radiography, NMR or ESR. For X-radiography, suitablelabels include radioisotopes such as barium or cesium, which emitdetectable radiation but are not overtly harmful to the subject.Suitable markers for NMR and ESR include those with a detectablecharacteristic spin, such as deuterium, which may be incorporated intothe antibody by labeling of nutrients for the relevant hybridoma.

[0336] A protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously, orintraperitoneally) into the mammal. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of 99 mTc. The labeled antibody or antibody fragment willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).)

[0337] Thus, the invention provides a diagnostic method of a disorder,which involves (a) assaying the expression of follistatin-3 polypeptidein cells or body fluid of an individual; (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed follistatin-3 polypeptide gene expression levelcompared to the standard expression level is indicative of a disorder.

[0338] Moreover, follistatin-3 polypeptides can be used to treatdisease. For example, patients can be administered follistatin-3polypeptides in an effort to replace absent or decreased levels of thefollistatin-3 polypeptide (e.g., insulin), to supplement absent ordecreased levels of a different polypeptide (e.g., hemoglobin S forhemoglobin B), to inhibit the activity of a polypeptide (e.g., anoncogene), to activate the activity of a polypeptide (e.g., by bindingto a receptor), to reduce the activity of a membrane bound receptor bycompeting with it for free ligand (e.g., soluble TNF receptors used inreducing inflammation), or to bring about a desired response (e.g.,blood vessel growth).

[0339] Similarly, antibodies directed to follistatin-3 polypeptides canalso be used to treat disease. For example, administration of anantibody directed to a follistatin-3 polypeptide can bind and reduceoverproduction of the polypeptide. Similarly, administration of anantibody can activate the polypeptide, such as by binding to apolypeptide bound to a membrane (receptor).

[0340] At the very least, the follistatin-3 polypeptides can be used asmolecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. Follistatin-3 polypeptides can also be used to raise antibodies,which in turn are used to measure protein expression from a recombinantcell, as a way of assessing transformation of the host cell. Moreover,follistatin-3 polypeptides can be used to test the following biologicalactivities.

[0341] Gene Therapy Methods.

[0342] Another aspect of the present invention is to gene therapymethods for treating disorders, diseases and conditions. The genetherapy methods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofthe follistatin-3 polypeptide of the present invention. This methodrequires a polynucleotide which codes for a follistatin-3 polypeptideoperatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO90/11092, which is herein incorporated by reference.

[0343] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to afollistatin-3 polynucleotide ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun, A., etal., J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al.,Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J.Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60:221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990);Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato,L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al.,Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated byreference. In one embodiment, the cells which are engineered arearterial cells. The arterial cells may be reintroduced into the patientthrough direct injection to the artery, the tissues surrounding theartery, or through catheter injection.

[0344] As discussed in more detail below, the follistatin-3polynucleotide constructs can be delivered by any method that deliversinjectable materials to the cells of an animal, such as, injection intothe interstitial space of tissues (heart, muscle, skin, lung, liver, andthe like). The follistatin-3 polynucleotide constructs may be deliveredin a pharmaceutically acceptable liquid or aqueous carrier.

[0345] In one embodiment, the follistatin-3 polynucleotide is deliveredas a naked polynucleotide. The term “naked” polynucleotide, DNA or RNArefers to sequences that are free from any delivery vehicle that acts toassist, promote or facilitate entry into the cell, including viralsequences, viral particles, liposome formulations, lipofectin orprecipitating agents and the like. However, the follistatin-3polynucleotides can also be delivered in liposome formulations andlipofectin formulations and the like can be prepared by methods wellknown to those skilled in the art. Such methods are described, forexample, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, whichare herein incorporated by reference.

[0346] The follistatin-3 polynucleotide vector constructs used in thegene therapy method are preferably constructs that will not integrateinto the host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

[0347] Any strong promoter known to those skilled in the art can be usedfor driving the expression of follistatin-3 DNA. Suitable promotersinclude adenoviral promoters, such as the adenoviral major latepromoter; or heterologous promoters, such as the cytomegalovirus (CMV)promoter; the respiratory syncytial virus (RSV) promoter; induciblepromoters, such as the MMT promoter, the metallothionein promoter; heatshock promoters; the albumin promoter; the ApoA1 promoter; human globinpromoters; viral thymidine kinase promoters, such as the Herpes Simplexthymidine kinase promoter; retroviral LTRs; the b-actin promoter; andhuman growth hormone promoters. The promoter also may be the nativepromoter for follistatin-3.

[0348] Unlike other gene therapy techniques, one major advantage ofintroducing naked nucleic acid sequences into target cells is thetransitory nature of the polynucleotide synthesis in the cells. Studieshave shown that non-replicating DNA sequences can be introduced intocells to provide production of the desired polypeptide for periods of upto six months.

[0349] The follistatin-3 polynucleotide construct can be delivered tothe interstitial space of tissues within the an animal, including ofmuscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart,lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[0350] For the naked acid sequence injection, an effective dosage amountof DNA or RNA will be in the range of from about 0.05 mg/kg body weightto about 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

[0351] The preferred route of administration is by the parenteral routeof injection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked follistatin-3DNA constructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

[0352] The naked polynucleotides are delivered by any method known inthe art, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, and so-called “gene guns”. These delivery methods are known inthe art.

[0353] As is evidenced in the Examples, naked follistatin-3 nucleic acidsequences can be administered in vivo results in the successfulexpression of follistatin-3 polypeptide in the femoral arteries ofrabbits.

[0354] The constructs may also be delivered with delivery vehicles suchas viral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

[0355] In certain embodiments, the follistatin-3 polynucleotideconstructs are complexed in a liposome preparation. Liposomalpreparations for use in the instant invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations. However, cationic liposomes are particularly preferredbecause a tight charge complex can be formed between the cationicliposome and the polyanionic nucleic acid. Cationic liposomes have beenshown to mediate intracellular delivery of plasmid DNA (Felgner et al.,Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is hereinincorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci.USA (1989) 86:6077-6081, which is herein incorporated by reference); andpurified transcription factors (Debs et al., J. Biol. Chem. (1990)265:10189-10192, which is herein incorporated by reference), infunctional form.

[0356] Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated byreference). Other commercially available liposomes include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boehringer).

[0357] Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

[0358] Similarly, anionic and neutral liposomes are readily available,such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easilyprepared using readily available materials. Such materials includephosphatidyl, choline, cholesterol, phosphatidyl ethanolamine,dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol(DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. Thesematerials can also be mixed with the DOTMA and DOTAP starting materialsin appropriate ratios. Methods for making liposomes using thesematerials are well known in the art.

[0359] For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15 EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

[0360] The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²+-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

[0361] Generally, the ratio of DNA to liposomes will be from about 10:1to about 1:10. Preferably, the ration will be from about 5:1 to about1:5. More preferably, the ration will be about 3:1 to about 1:3. Stillmore preferably, the ratio will be about 1:1.

[0362] U.S. Pat. No. 5,676,954 (which is herein incorporated byreference) reports on the injection of genetic material, complexed withcationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355,4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide cationic lipids for use intransfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466,5,693,622, 5,580,859, 5,703,055, and international publication no. WO94/9469 (which are herein incorporated by reference) provide methods fordelivering DNA-cationic lipid complexes to mammals.

[0363] In certain embodiments, cells are be engineered, ex vivo or invivo, using a retroviral particle containing RNA which comprises asequence encoding follistatin-3. Retroviruses from which the retroviralplasmid vectors may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammarytumor virus.

[0364] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy 1:5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0365] The producer cell line generates infectious retroviral vectorparticles which include polynucleotide encoding follistatin-3. Suchretroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express follistatin-3.

[0366] In certain other embodiments, cells are engineered, ex vivo or invivo, with follistatin-3 polynucleotide contained in an adenovirusvector. Adenovirus can be manipulated such that it encodes and expressesfollistatin-3, and at the same time is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. Adenovirusexpression is achieved without integration of the viral DNA into thehost cell chromosome, thereby alleviating concerns about insertionalmutagenesis. Furthermore, adenoviruses have been used as live entericvaccines for many years with an excellent safety profile (Schwartz, A.R. et al. (1974) Am. Rev. Respir. Dis.109:233-238). Finally, adenovirusmediated gene transfer has been demonstrated in a number of instancesincluding transfer of alpha-1-antitrypsin and CFTR to the lungs ofcotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434;Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green, M. et al. (1979) Proc. Natl.Acad. Sci. USA 76:6606).

[0367] Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

[0368] Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, for example, the HARP promoter of the present invention, butcannot replicate in most cells. Replication deficient adenoviruses maybe deleted in one or more of all or a portion of the following genes:E1a, E1b, E3, E4, E2a, or L1 through L5.

[0369] In certain other embodiments, the cells are engineered, ex vivoor in vivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

[0370] For example, an appropriate AAV vector for use in the presentinvention will include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The follistatin-3polynucleotide construct is inserted into the AAV vector using standardcloning methods, such as those found in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press (1989). Therecombinant AAV vector is then transfected into packaging cells whichare infected with a helper virus, using any standard technique,including lipofection, electroporation, calcium phosphate precipitation,etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses,vaccinia viruses, or herpes viruses. Once the packaging cells aretransfected and infected, they will produce infectious AAV viralparticles which contain the follistatin-3 polynucleotide construct.These viral particles are then used to transduce eukaryotic cells,either ex vivo or in vivo. The transduced cells will contain thefollistatin-3 polynucleotide construct integrated into its genome, andwill express follistatin-3.

[0371] Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding follistatin-3) via homologous recombination (see, e.g.,U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International PublicationNo. WO 96/29411, published Sep. 26, 1996; International Publication No.WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad.Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438(1989). This method involves the activation of a gene which is presentin the target cells, but which is not normally expressed in the cells,or is expressed at a lower level than desired.

[0372] Polynucleotide constructs are made, using standard techniquesknown in the art, which contain the promoter with targeting sequencesflanking the promoter. Suitable promoters are described herein. Thetargeting sequence is sufficiently complementary to an endogenoussequence to permit homologous recombination of the promoter-targetingsequence with the endogenous sequence. The targeting sequence will besufficiently near the 5′ end of the follistatin-3 desired endogenouspolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination.

[0373] The promoter and the targeting sequences can be amplified usingPCR. Preferably, the amplified promoter contains distinct restrictionenzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

[0374] The promoter-targeting sequence construct is delivered to thecells, either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

[0375] The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous follistatin-3 sequence isplaced under the control of the promoter. The promoter then drives theexpression of the endogenous follistatin-3 sequence.

[0376] The polynucleotides encoding follistatin-3 may be administeredalong with other polynucleotides encoding other angiongenic proteins.Angiogenic proteins include, but are not limited to, acidic and basicfibroblast growth factors, VEGF-1, epidermal growth factor alpha andbeta, platelet-derived endothelial cell growth factor, platelet-derivedgrowth factor, tumor necrosis factor alpha, hepatocyte growth factor,insulin like growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

[0377] Preferably, the polynucleotide encoding follistatin-3 contains asecretory signal sequence that facilitates secretion of the protein.Typically, the signal sequence is positioned in the coding region of thepolynucleotide to be expressed towards or at the 5′ end of the codingregion. The signal sequence may be homologous or heterologous to thepolynucleotide of interest and may be homologous or heterologous to thecells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

[0378] Any mode of administration of any of the above-describedpolynucleotides constructs can be used so long as the mode results inthe expression of one or more molecules in an amount sufficient toprovide a therapeutic effect. This includes direct needle injection,systemic injection, catheter infusion, biolistic injectors, particleaccelerators (i.e., “gene guns”), gelfoam sponge depots, othercommercially available depot materials, osmotic pumps (e.g., Alzaminipumps), oral or suppositorial solid (tablet or pill) pharmaceuticalformulations, and decanting or topical applications during surgery. Forexample, direct injection of naked calcium phosphate-precipitatedplasmid into rat liver and rat spleen or a protein-coated plasmid intothe portal vein has resulted in gene expression of the foreign gene inthe rat livers (Kaneda et al., Science 243:375 (1989)).

[0379] A preferred method of local administration is by directinjection. Preferably, a recombinant molecule of the present inventioncomplexed with a delivery vehicle is administered by direct injectioninto or locally within the area of arteries. Administration of acomposition locally within the area of arteries refers to injecting thecomposition centimeters and preferably, millimeters within arteries.

[0380] Another method of local administration is to contact apolynucleotide construct of the present invention in or around asurgical wound. For example, a patient can undergo surgery and thepolynucleotide construct can be coated on the surface of tissue insidethe wound or the construct can be injected into areas of tissue insidethe wound.

[0381] Therapeutic compositions useful in systemic administration,include recombinant molecules of the present invention complexed to atargeted delivery vehicle of the present invention. Suitable deliveryvehicles for use with systemic administration comprise liposomescomprising ligands for targeting the vehicle to a particular site.

[0382] Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

[0383] Determining an effective amount of substance to be delivered candepend upon a number of factors including, for example, the chemicalstructure and biological activity of the substance, the age and weightof the animal, the precise condition requiring treatment and itsseverity, and the route of administration. The frequency of treatmentsdepends upon a number of factors, such as the amount of polynucleotideconstructs administered per dose, as well as the health and history ofthe subject. The precise amount, number of doses, and timing of doseswill be determined by the attending physician or veterinarian.

[0384] Therapeutic compositions of the present invention can beadministered to any animal, preferably to mammals and birds. Preferredmammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle,horses and pigs, with humans being particularly preferred.

[0385] Biological Activities of Follistatin-3.

[0386] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, can be used in assays to test for one ormore biological activities. If follistatin-3 polynucleotides orpolypeptides, or agonists or antagonists of follistatin-3, do exhibitactivity in a particular assay, it is likely that follistatin-3 may beinvolved in the diseases associated with the biological activity.Therefore, follistatin-3 could be used to treat the associated disease.

[0387] Immune Activity

[0388] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3 may be useful in treating deficiencies ordisorders of the immune system, by activating or inhibiting theproliferation, differentiation, or mobilization (chemotaxis) of immunecells. Immune cells develop through a process called hematopoiesis,producing myeloid (platelets, red blood cells, neutrophils, andmacrophages) and lymphoid (B and T lymphocytes) cells from pluripotentstem cells. The etiology of these immune deficiencies or disorders maybe genetic, somatic, such as cancer or some autoimmune disorders,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover,follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, can be used as a marker or detector of aparticular immune system disease or disorder.

[0389] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, may be useful in treating or detectingdeficiencies or disorders of hematopoietic cells. Follistatin-3polynucleotides or polypeptides, or agonists or antagonists offollistatin-3, could be used to increase differentiation andproliferation of hematopoietic cells, including the pluripotent stemcells, in an effort to treat those disorders associated with a decreasein certain (or many) types hematopoietic cells. Examples of immunologicdeficiency syndromes include, but are not limited to: blood proteindisorders (e.g. agammaglobulinemia, dysgammaglobulinemia), ataxiatelangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIVinfection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome,lymphopenia, phagocyte bactericidal dysfunction, severe combinedimmunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia,thrombocytopenia, or hemoglobinuria.

[0390] Moreover, follistatin-3 polynucleotides or polypeptides, oragonists or antagonists of follistatin-3, can also be used to modulatehemostatic (the stopping of bleeding) or thrombolytic activity (clotformation). For example, by increasing hemostatic or thrombolyticactivity, follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, could be used to treat blood coagulationdisorders (e.g., afibrinogenemia, factor deficiencies), blood plateletdisorders (e.g. thrombocytopenia), or wounds resulting from trauma,surgery, or other causes. Alternatively, follistatin-3 polynucleotidesor polypeptides, or agonists or antagonists of follistatin-3, that candecrease hemostatic or thrombolytic activity could be used to inhibit ordissolve clotting, important in the treatment of heart attacks(infarction), strokes, or scarring.

[0391] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, may also be useful in treating ordetecting autoimmune disorders. Many autoimmune disorders result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration offollistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, that can inhibit an immune response,particularly the proliferation, differentiation, or chemotaxis ofT-cells, may be an effective therapy in preventing autoimmune disorders.

[0392] Examples of autoimmune disorders that can be treated or detectedinclude, but are not limited to: Addison's Disease, hemolytic anemia,antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergicencephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves'Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter'sDisease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic LupusErythematosus, Autoimmune Pulmonary Inflammation, Guillain-BarreSyndrome, insulin dependent diabetes mellitis, and autoimmuneinflammatory eye disease.

[0393] Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by follistatin-3 polynucleotides or polypeptides, or agonistsor antagonists of follistatin-3. Moreover, these molecules can be usedto treat anaphylaxis, hypersensitivity to an antigenic molecule, orblood group incompatibility.

[0394] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, may also be used to treat and/or preventorgan rejection or graft-versus-host disease (GVHD). Organ rejectionoccurs by host immune cell destruction of the transplanted tissuethrough an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. The administration of follistatin-3polynucleotides or polypeptides, or agonists or antagonists offollistatin-3, that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing organ rejection or GVHD.

[0395] Similarly, follistatin-3 polynucleotides or polypeptides, oragonists or antagonists of follistatin-3, may also be used to modulateinflammation. For example, follistatin-3 polynucleotides orpolypeptides, or agonists or antagonists of follistatin-3, may inhibitthe proliferation and differentiation of cells involved in aninflammatory response. These molecules can be used to treat inflammatoryconditions, both chronic and acute conditions, including inflammationassociated with infection (e.g., septic shock, sepsis, or systemicinflammatory response syndrome (SIRS)), ischemia-reperfusion injury,endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine induced lung injury,inflammatory bowel disease, Crohn's disease, or resulting from overproduction of cytokines (e.g., TNF or IL-1.)

[0396] Hyperproliferative Disorders.

[0397] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, can be used to treat or detecthyperproliferative disorders, including neoplasms. Follistatin-3polynucleotides or polypeptides, or agonists or antagonists offollistatin-3, may inhibit the proliferation of the disorder throughdirect or indirect interactions. Alternatively, follistatin-3polynucleotides or polypeptides, or agonists or antagonists offollistatin-3, may proliferate other cells which can inhibit thehyperproliferative disorder.

[0398] For example, by increasing an immune response, particularlyincreasing antigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

[0399] Examples of hyperproliferative disorders that can be treated ordetected by follistatin-3 polynucleotides or polypeptides, or agonistsor antagonists of follistatin-3, include, but are not limited toneoplasms located in the: abdomen, bone, breast, digestive system,liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid,pituitary, testicles, ovary, thymus, thyroid), eye, head and neck,nervous (central and peripheral), lymphatic system, pelvic, skin, softtissue, spleen, thoracic, and urogenital.

[0400] Similarly, other hyperproliferative disorders can also be treatedor detected by follistatin-3 polynucleotides or polypeptides, oragonists or antagonists of follistatin-3. Examples of suchhyperproliferative disorders include, but are not limited to:hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,Gaucher's Disease, histiocytosis, and any other hyperproliferativedisease, besides neoplasia, located in an organ system listed above.

[0401] Cardiovascular Disorders.

[0402] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, encoding follistatin-3 may be used totreat cardiovascular disorders, including peripheral artery disease,such as limb ischemia.

[0403] Cardiovascular disorders include cardiovascular abnormalities,such as arterio-arterial fistula, arteriovenous fistula, cerebralarteriovenous malformations, congenital heart defects, pulmonaryatresia, and Scimitar Syndrome. Congenital heart defects include aorticcoarctation, cor triatriatum, coronary vessel anomalies, crisscrossheart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,Eisenmenger complex, hypoplastic left heart syndrome, levocardia,tetralogy of fallot, transposition of great vessels, double outlet rightventricle, tricuspid atresia, persistent truncus arteriosus, and heartseptal defects, such as aortopulmonary septal defect, endocardialcushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricularheart septal defects.

[0404] Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

[0405] Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

[0406] Heart valve disease include aortic valve insufficiency, aorticvalve stenosis, hear murmurs, aortic valve prolapse, mitral valveprolapse, tricuspid valve prolapse, mitral valve insufficiency, mitralvalve stenosis, pulmonary atresia, pulmonary valve insufficiency,pulmonary valve stenosis, tricuspid atresia, tricuspid valveinsufficiency, and tricuspid valve stenosis.

[0407] Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

[0408] Myocardial ischemias include coronary disease, such as anginapectoris, coronary aneurysm, coronary arteriosclerosis, coronarythrombosis, coronary vasospasm, myocardial infarction and myocardialstunning.

[0409] Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

[0410] Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

[0411] Arterial occlusive diseases include arteriosclerosis,intermittent claudication, carotid stenosis, fibromuscular dysplasias,mesenteric vascular occlusion, Moyamoya disease, renal arteryobstruction, retinal artery occlusion, and thromboangiitis obliterans.

[0412] Cerebrovascular disorders include carotid artery diseases,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebralartery diseases, cerebral embolism and thrombosis, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, cerebralhemorrhage, epidural hematoma, subdural hematoma, subaraxhnoidhemorrhage, cerebral infarction, cerebral ischemia (includingtransient), subclavian steal syndrome, periventricular leukomalacia,vascular headache, cluster headache, migraine, and vertebrobasilarinsufficiency.

[0413] Embolisms include air embolisms, amniotic fluid embolisms,cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonaryembolisms, and thromoboembolisms. Thrombosis include coronarythrombosis, hepatic vein thrombosis, retinal vein occlusion, carotidartery thrombosis, sinus thrombosis, Wallenberg's syndrome, andthrombophlebitis.

[0414] Ischemia includes cerebral ischemia, ischemic colitis,compartment syndromes, anterior compartment syndrome, myocardialischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitisincludes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

[0415] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, are especially effective for the treatmentof critical limb ischemia and coronary disease. As shown in theExamples, administration of follistatin-3 polynucleotides andpolypeptides to an experimentally induced ischemia rabbit hindlimb mayrestore blood pressure ratio, blood flow, angiographic score, andcapillary density.

[0416] Follistatin-3 polypeptides may be administered using any methodknown in the art, including, but not limited to, direct needle injectionat the delivery site, intravenous injection, topical administration,catheter infusion, biolistic injectors, particle accelerators, gelfoamsponge depots, other commercially available depot materials, osmoticpumps, oral or suppositorial solid pharmaceutical formulations,decanting or topical applications during surgery, aerosol delivery. Suchmethods are known in the art. Follistatin-3 polypeptides may beadministered as part of a pharmaceutical composition, described in moredetail below. Methods of delivering follistatin-3 polynucleotides aredescribed in more detail herein.

[0417] Angiogenesis.

[0418] The naturally occurring balance between endogenous stimulatorsand inhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

[0419] The present invention provides for treatment of diseases ordisorders associated with neovascularization by administration of thefollistatin-3 polynucleotides and/or polypeptides of the invention, aswell as agonists or antagonists of follistatin-3. Malignant andmetastatic conditions which can be treated with the polynucleotides andpolypeptides, or agonists or antagonists of the invention include, butare not limited to, malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)):

[0420] Ocular disorders associated with neovascularization which can betreated with the follistatin-3 polynucleotides and polypeptides of thepresent invention (including follistatin-3 agonists and/or antagonists)include, but are not limited to: neovascular glaucoma, diabeticretinopathy, retinoblastoma, retrolental fibroplasia, uveitis,retinopathy of prematurity macular degeneration, corneal graftneovascularization, as well as other eye inflammatory diseases, oculartumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

[0421] Additionally, disorders which can be treated with thefollistatin-3 polynucleotides and polypeptides of the present invention(including follistatin-3 agonist and/or antagonists) include, but arenot limited to, hemangioma, arthritis, psoriasis, angiofibroma,atherosclerotic plaques, delayed wound healing, granulations, hemophilicjoints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome,pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

[0422] Moreover, disorders and/or states, which can be treated with betreated with the follistatin-3 polynucleotides and polypeptides of thepresent invention (including follistatin-3 agonist and/or antagonists)include, but are not limited to, solid tumors, blood born tumors such asleukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, forexample hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenicdiseases, for example, diabetic retinopathy, retinopathy of prematurity,macular degeneration, corneal graft rejection, neovascular glaucoma,retrolental fibroplasia, rubcosis, retinoblastoma, and uvietis, delayedwound healing, endometriosis, vascluogenesis, granulations, hypertrophicscars (keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

[0423] Diseases at the Cellular Level.

[0424] Diseases associated with increased cell survival or theinhibition of apoptosis that could be treated or detected byfollistatin-3 polynucleotides or polypeptides, as well as antagonists oragonists of follistatin-3, include cancers (such as follicularlymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection. In preferred embodiments,follistatin-3 polynucleotides, polypeptides, and/or antagonists of theinvention are used to inhibit growth, progression, and/or metasis ofcancers, in particular those listed above.

[0425] Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by follistatin-3polynucleotides or polypeptides, or agonists or antagonists offollistatin-3, include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

[0426] Diseases associated with increased apoptosis that could betreated or detected by follistatin-3 polynucleotides or polypeptides, aswell as agonists or antagonists of follistatin-3, include AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellardegeneration and brain tumor or prior associated disease); autoimmunedisorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

[0427] Wound Healing and Epithelial Cell Proliferation.

[0428] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing follistatin-3 polynucleotidesor polypeptides, as well as agonists or antagonists of follistatin-3,for therapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.follistatin-3 polynucleotides or polypeptides, as well as agonists orantagonists of follistatin-3, may be clinically useful in stimulatingwound healing including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as uremia, malnutrition, vitamin deficiencies andcomplications associted with systemic treatment with steroids, radiationtherapy and antineoplastic drugs and antimetabolites. Follistatin-3polynucleotides or polypeptides, as well as agonists or antagonists offollistatin-3, could be used to promote dermal reestablishmentsubsequent to dermal loss

[0429] Follistatin-3 polynucleotides or polypeptides, as well asagonists or antagonists of follistatin-3, could be used to increase theadherence of skin grafts to a wound bed and to stimulatere-epithelialization from the wound bed. The following are types ofgrafts that follistatin-3 polynucleotides or polypeptides, agonists orantagonists of follistatin-3, could be used to increase adherence to awound bed: autografts, artificial skin, allografts, autodermic graft,autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft,brephoplastic grafts, cutis graft, delayed graft, dermic graft,epidermic graft, fascia graft, full thickness graft, heterologous graft,xenograft, homologous graft, hyperplastic graft, lamellar graft, meshgraft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft,pedicle graft, penetrating graft, split skin graft, thick split graft.follistatin-3 polynucleotides or polypeptides, as well as agonists orantagonists of follistatin-3, can be used to promote skin strength andto improve the appearance of aged skin.

[0430] It is believed that follistatin-3 polynucleotides orpolypeptides, as well as agonists or antagonists of follistatin-3, willalso produce changes in hepatocyte proliferation, and epithelial cellproliferation in the lung, breast, pancreas, stomach, small intesting,and large intestine. Follistatin-3 polynucleotides or polypeptides, aswell as agonists or antagonists of follistatin-3, could promoteproliferation of epithelial cells such as sebocytes, hair follicles,hepatocytes, type II pneumocytes, mucin-producing goblet cells, andother epithelial cells and their progenitors contained within the skin,lung, liver, and gastrointestinal tract. Follistatin-3 polynucleotidesor polypeptides, agonists or antagonists of follistatin-3, may promoteproliferation of endothelial cells, keratinocytes, and basalkeratinocytes.

[0431] Follistatin-3 polynucleotides or polypeptides, as well asagonists or antagonists of follistatin-3, could also be used to reducethe side effects of gut toxicity that result from radiation,chemotherapy treatments or viral infections. Follistatin-3polynucleotides or polypeptides, as well as agonists or antagonists offollistatin-3, may have a cytoprotective effect on the small intestinemucosa. Follistatin-3 polynucleotides or polypeptides, as well asagonists or antagonists of follistatin-3, may also stimulate healing ofmucositis (mouth ulcers) that result from chemotherapy and viralinfections.

[0432] Follistatin-3 polynucleotides or polypeptides, as well asagonists or antagonists of follistatin-3, could further be used in fullregeneration of skin in full and partial thickness skin defects,including bums, (i.e., repopulation of hair follicles, sweat glands, andsebaceous glands), treatment of other skin defects such as psoriasis.Follistatin-3 polynucleotides or polypeptides, as well as agonists orantagonists of follistatin-3, could be used to treat epidermolysisbullosa, a defect in adherence of the epidermis to the underlying dermiswhich results in frequent, open and painful blisters by acceleratingreepithelialization of these lesions. Follistatin-3 polynucleotides orpolypeptides, as well as agonists or antagonists of follistatin-3, couldalso be used to treat gastric and doudenal ulcers and help heal by scarformation of the mucosal lining and regeneration of glandular mucosa andduodenal mucosal lining more rapidly. Inflamamatory bowel diseases, suchas Crohn's disease and ulcerative colitis, are diseases which result indestruction of the mucosal surface of the small or large intestine,respectively. Thus, follistatin-3 polynucleotides or polypeptides, aswell as agonists or antagonists of follistatin-3, could be used topromote the resurfacing of the mucosal surface to aid more rapid healingand to prevent progression of inflammatory bowel disease. Treatment withfollistatin-3 polynucleotides or polypeptides, agonists or antagonistsof follistatin-3, is expected to have a significant effect on theproduction of mucus throughout the gastrointestinal tract and could beused to protect the intestinal mucosa from injurious substances that areingested or following surgery. Follistatin-3 polynucleotides orpolypeptides, as well as agonists or antagonists of follistatin-3, couldbe used to treat diseases associate with the under expression offollistatin-3.

[0433] Moreover, follistatin-3 polynucleotides or polypeptides, as wellas agonists or antagonists of follistatin-3, could be used to preventand heal damage to the lungs due to various pathological states. Agrowth factor such as follistatin-3 polynucleotides or polypeptides, aswell as agonists or antagonists of follistatin-3, which could stimulateproliferation and differentiation and promote the repair of alveoli andbrochiolar epithelium to prevent or treat acute or chronic lung damage.For example, emphysema, which results in the progressive loss of aveoli,and inhalation injuries, i.e., resulting from smoke inhalation andburns, that cause necrosis of the bronchiolar epithelium and alveolicould be effectively treated using follistatin-3 polynucleotides orpolypeptides, agonists or antagonists of follistatin-3. Also,follistatin-3 polynucleotides or polypeptides, as well as agonists orantagonists of follistatin-3, could be used to stimulate theproliferation of and differentiation of type II pneumocytes, which mayhelp treat or prevent disease such as hyaline membrane diseases, such asinfant respiratory distress syndrome and bronchopulmonary displasia, inpremature infants.

[0434] Follistatin-3 polynucleotides or polypeptides, as well asagonists or antagonists of follistatin-3, could stimulate theproliferation and differentiation of hepatocytes and, thus, could beused to alleviate or treat liver diseases and pathologies such asfulminant liver failure caused by cirrhosis, liver damage caused byviral hepatitis and toxic substances (i.e., acetaminophen, carbontetraholoride and other hepatotoxins known in the art).

[0435] In addition, follistatin-3 polynucleotides or polypeptides, aswell as agonists or antagonists of follistatin-3, could be used treat orprevent the onset of diabetes mellitus. In patients with newly diagnosedTypes I and II diabetes, where some islet cell function remains,follistatin-3 polynucleotides or polypeptides, as well as agonists orantagonists of follistatin-3, could be used to maintain the isletfunction so as to alleviate, delay or prevent permanent manifestation ofthe disease. Also, follistatin-3 polynucleotides or polypeptides, aswell as agonists or antagonists of follistatin-3, could be used as anauxiliary in islet cell transplantation to improve or promote islet cellfunction.

[0436] Infectious Disease.

[0437] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, can be used to treat or detect infectiousagents. For example, by increasing the immune response, particularlyincreasing the proliferation and differentiation of B and/or T cells,infectious diseases may be treated. The immune response may be increasedby either enhancing an existing immune response, or by initiating a newimmune response. Alternatively, follistatin-3 polynucleotides orpolypeptides, or agonists or antagonists of follistatin-3, may alsodirectly inhibit the infectious agent, without necessarily eliciting animmune response.

[0438] Viruses are one example of an infectious agent that can causedisease or symptoms that can be treated or detected by follistatin-3polynucleotides or polypeptides, or agonists or antagonists offollistatin-3. Examples of viruses, include, but are not limited to thefollowing DNA and RNA viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis),Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae,Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae(e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), andTogaviridae (e.g., Rubivirus). Viruses falling within these families cancause a variety of diseases or symptoms, including, but not limited to:arthritis, bronchiollitis, encephalitis, eye infections (e.g.,conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B,C, E, Chronic Active, Delta), meningitis, opportunistic infections(e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagicfever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio,leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g.,Kaposi's, warts), and viremia. Follistatin-3 polynucleotides orpolypeptides, or agonists or antagonists of follistatin-3, can be usedto treat or detect any of these symptoms or diseases.

[0439] Similarly, bacterial or fungal agents that can cause disease orsymptoms and that can be treated or detected by follistatin-3polynucleotides or polypeptides, or agonists or antagonists offollistatin-3, include, but not limited to, the following Gram-Negativeand Gram-positive bacterial families and fungi: Actinomycetales (e.g.,Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae(e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis,Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella,Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter,Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae(e.g., Acinetobacter, Gonorrhea, Menigococcal), PasteurellaceaInfections (e.g., Actinobacillus, Heamophilus, Pasteurella),Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, andStaphylococcal. These bacterial or fungal families can cause thefollowing diseases or symptoms, including, but not limited to:bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis,uveitis), gingivitis, opportunistic infections (e.g., AIDS relatedinfections), paronychia, prosthesis-related infections, Reiter'sDisease, respiratory tract infections, such as Whooping Cough orEmpyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery,Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea,meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Follistatin-3 polynucleotides or polypeptides, oragonists or antagonists of follistatin-3, can be used to treat or detectany of these symptoms or diseases.

[0440] Moreover, parasitic agents causing disease or symptoms that canbe treated or detected by follistatin-3 polynucleotides or polypeptides,or agonists or antagonists of follistatin-3, include, but not limitedto, the following families: Amebiasis, Babesiosis, Coccidiosis,Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis,Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas. These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,giardiasis), liver disease, lung disease, opportunistic infections(e.g., AIDS related), Malaria, pregnancy complications, andtoxoplasmosis. Follistatin-3 polynucleotides or polypeptides, oragonists or antagonists of follistatin-3, can be used to treat or detectany of these symptoms or diseases.

[0441] Preferably, treatment using follistatin-3 polynucleotides orpolypeptides, or agonists or antagonists of follistatin-3, could eitherbe by administering an effective amount of follistatin-3 polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith follistatin-3 polynucleotide, and returning the engineered cells tothe patient (ex vivo therapy). Moreover, the follistatin-3 polypeptideor polynucleotide can be used as an antigen in a vaccine to raise animmune response against infectious disease.

[0442] Regeneration.

[0443] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, can be used to differentiate, proliferate,and attract cells, leading to the regeneration of tissues. (See, Science276:59-87 (1997).) The regeneration of tissues could be used to repair,replace, or protect tissue damaged by congenital defects, trauma(wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis,osteocarthritis, periodontal disease, liver failure), surgery, includingcosmetic plastic surgery, fibrosis, reperfusion injury, or systemiccytokine damage.

[0444] Tissues that could be regenerated using the present inventioninclude organs (e.g., pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

[0445] Moreover, follistatin-3 polynucleotides or polypeptides, oragonists or antagonists of follistatin-3, may increase regeneration oftissues difficult to heal. For example, increased tendon/ligamentregeneration would quicken recovery time after damage. Follistatin-3polynucleotides or polypeptides, or agonists or antagonists offollistatin-3,of the present invention could also be usedprophylactically in an effort to avoid damage. Specific diseases thatcould be treated include of tendinitis, carpal tunnel syndrome, andother tendon or ligament defects. A further example of tissueregeneration of non-healing wounds includes pressure ulcers, ulcersassociated with vascular insufficiency, surgical, and traumatic wounds.

[0446] Similarly, nerve and brain tissue could also be regenerated byusing follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, to proliferate and differentiate nervecells. Diseases that could be treated using this method include centraland peripheral nervous system diseases, neuropathies, or mechanical andtraumatic disorders (e.g., spinal cord disorders, head trauma,cerebrovascular disease, and stoke). Specifically, diseases associatedwith peripheral nerve injuries, peripheral neuropathy (e.g., resultingfrom chemotherapy or other medical therapies), localized neuropathies,and central nervous system diseases (e.g., Alzheimer's disease,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome), could all be treated using thefollistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3.

[0447] Chemotaxis.

[0448] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, may have chemotaxis activity. A chemotaxicmolecule attracts or mobilizes cells (e.g., monocytes, fibroblasts,neutrophils, T-cells, mast cells, eosinophils, epithelial and/orendothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

[0449] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, may increase chemotaxic activity ofparticular cells. These chemotactic molecules can then be used to treatinflammation, infection, hyperproliferative disorders, or any immunesystem disorder by increasing the number of cells targeted to aparticular location in the body. For example, chemotaxic molecules canbe used to treat wounds and other trauma to tissues by attracting immunecells to the injured location. As a chemotactic molecule, follistatin-3could also attract fibroblasts, which can be used to treat wounds.

[0450] It is also contemplated that follistatin-3 polynucleotides orpolypeptides, or agonists or antagonists of follistatin-3, may inhibitchemotactic activity. These molecules could also be used to treatdisorders. Thus, follistatin-3 polynucleotides or polypeptides, oragonists or antagonists of follistatin-3, could be used as an inhibitorof chemotaxis.

[0451] Binding Activity.

[0452] Follistatin-3 polypeptides may be used to screen for moleculesthat bind to follistatin-3 or for molecules to which follistatin-3binds. The binding of follistatin-3 and the molecule may activate(agonist), increase, inhibit (antagonist), or decrease activity of thefollistatin-3 or the molecule bound. Examples of such molecules includeantibodies, oligonucleotides, proteins (e.g., receptors), or smallmolecules.

[0453] Preferably, the molecule is closely related to the natural ligandof follistatin-3, e.g., a fragment of the ligand, or a naturalsubstrate, a ligand, a structural or functional mimetic (See, Coligan etal., Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly,the molecule can be closely related to the natural receptor to whichfollistatin-3 binds, or at least, a fragment of the receptor capable ofbeing bound by follistatin-3 (e.g., active site). In either case, themolecule can be rationally designed using known techniques.

[0454] Preferably, the screening for these molecules involves producingappropriate cells which express follistatin-3, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing follistatin-3(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding stimulation, or inhibition of activity ofeither follistatin-3 or the molecule.

[0455] The assay may simply test binding of a candidate compound tofollistatin-3, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to follistatin-3.

[0456] Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining follistatin-3, measuring follistatin-3/molecule activity orbinding, and comparing the follistatin-3/molecule activity or binding toa standard.

[0457] Preferably, an ELISA assay can measure follistatin-3 level oractivity in a sample (e.g., biological sample) using a monoclonal orpolyclonal antibody. The antibody can measure follistatin-3 level oractivity by either binding, directly or indirectly, to follistatin-3 orby competing with follistatin-3 for a substrate.

[0458] Additionally, the receptor to which follistatin-3 binds can beidentified by numerous methods known to those of skill in the art, forexample, ligand panning and FACS sorting (Coligan, et al., CurrentProtocols in Immun., 1(2), Chapter 5, (1991)). For example, expressioncloning is employed wherein polyadenylated RNA is prepared from a cellresponsive to the polypeptides, for example, NIH3T3 cells which areknown to contain multiple receptors for the FGF family proteins, andSC-3 cells, and a cDNA library created from this RNA is divided intopools and used to transfect COS cells or other cells that are notresponsive to the polypeptides. Transfected cells which are grown onglass slides are exposed to the polypeptide of the present invention,after they have been labelled. The polypeptides can be labeled by avariety of means including iodination or inclusion of a recognition sitefor a site-specific protein kinase.

[0459] Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

[0460] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to X-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[0461] Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of follistatin-3thereby effectively generating agonists and antagonists offollistatin-3. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238,5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol.16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76(1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13(1998) (each of these patents and publications are hereby incorporatedby reference). In one embodiment, alteration of follistatin-3polynucleotides and corresponding polypeptides may be achieved by DNAshuffling. DNA shuffling involves the assembly of two or more DNAsegments into a desired follistatin-3 molecule by homologous, orsite-specific, recombination. In another embodiment, follistatin-3polynucleotides and corresponding polypeptides may be alterred by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of follistatin-3 may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules. In preferred embodiments, the heterologousmolecules are follistatin-3 family members. In further preferredembodiments, the heterologous molecule is a growth factor such as, forexample, platelet-derived growth factor (PDGF), insulin-like growthfactor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growthfactor (EGF), fibroblast growth factor (FGF), TGF-beta, bonemorphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins Aand B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiationfactors (GDFs), nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2,TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).

[0462] Other preferred fragments are biologically active follistatin-3fragments. Biologically active fragments are those exhibiting activitysimilar, but not necessarily identical, to an activity of thefollistatin-3 polypeptide. The biological activity of the fragments mayinclude an improved desired activity, or a decreased undesirableactivity.

[0463] Additionally, this invention provides a method of screeningcompounds to identify those which modulate the action of the polypeptideof the present invention. An example of such an assay comprisescombining a mammalian fibroblast cell, a the polypeptide of the presentinvention, the compound to be screened and ³[H] thymidine under cellculture conditions where the fibroblast cell would normally proliferate.A control assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of [³H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of [³H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

[0464] In another method, a mammalian cell or membrane preparationexpressing a receptor for a polypeptide of the present invention isincubated with a labeled polypeptide of the present invention in thepresence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction of acompound to be screened and the follistatin-3 receptor is measured andthe ability of the compound to bind to the receptor and elicit a secondmessenger response is measured to determine if the compound is apotential agonist or antagonist. Such second messenger systems includebut are not limited to, cAMP guanylate cyclase, ion channels orphosphoinositide hydrolysis.

[0465] All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting thefollistatin-3/molecule. Moreover, the assays can discover agents whichmay inhibit or enhance the production of follistatin-3 from suitablymanipulated cells or tissues.

[0466] Therefore, the invention includes a method of identifyingcompounds which bind to follistatin-3 comprising the steps of: (a)incubating a candidate binding compound with follistatin-3; and (b)determining if binding has occurred. Moreover, the invention includes amethod of identifying agonists/antagonists comprising the steps of: (a)incubating a candidate compound with follistatin-3, (b) assaying abiological activity, and (b) determining if a biological activity offollistatin-3 has been altered.

[0467] Antisense And Ribozyme (Antagonists).

[0468] In specific embodiments, antagonists according to the presentinvention are nucleic acids corresponding to the sequences contained inSEQ ID NO:1, or the complementary strand thereof, and/or to nucleotidesequences contained in the deposited clone follistatin-3. In oneembodiment, antisense sequence is generated internally by the organism,in another embodiment, the antisense sequence is separately administered(see, for example, O'Connor, J., Neurochem. 56:560 (1991).Oligodeoxynucleotides as Anitsense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Antisense technology can be used tocontrol gene expression through antisense DNA or RNA, or throughtriple-helix formation. Antisense techniques are discussed for example,in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Triple helix formation is discussed in, for instance, Lee etal., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methodsare based on binding of a polynucleotide to a complementary DNA or RNA.

[0469] For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

[0470] In one embodiment, the follistatin-3 antisense nucleic acid ofthe invention is produced intracellularly by transcription from anexogenous sequence. For example, a vector or a portion thereof, istranscribed, producing an antisense nucleic acid (RNA) of the invention.Such a vector would contain a sequence encoding the follistatin-3antisense nucleic acid. Such a vector can remain episomal or becomechromosomally integrated, as long as it can be transcribed to producethe desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others know in the art, used for replication andexpression in vertebrate cells. Expression of the sequence encodingfollistatin-3, or fragments thereof, can be by any promoter known in theart to act in vertebrate, preferably human cells. Such promoters can beinducible or constitutive. Such promoters include, but are not limitedto, the SV40 early promoter region (Bernoist and Chambon, Nature29:304-310 (1981), the promoter contained in the 3′ long terminal repeatof Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), theherpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.78:1441-1445 (1981), the regulatory sequences of the metallothioneingene (Brinster, et al., Nature 296:39-42 (1982)), etc.

[0471] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of afollistatin-3 gene. However, absolute complementarity, althoughpreferred, is not required. A sequence “complementary to at least aportion of an RNA,” referred to herein, means a sequence havingsufficient complementarity to be able to hybridize with the RNA, forminga stable duplex; in the case of double stranded follistatin-3 antisensenucleic acids, a single strand of the duplex DNA may thus be tested, ortriplex formation may be assayed. The ability to hybridize will dependon both the degree of complementarity and the length of the antisensenucleic acid Generally, the larger the hybridizing nucleic acid, themore base mismatches with a follistatin-3 RNA it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

[0472] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of follistatin-3 shown in FIGS.1A, 1B, and 1C could be used in an antisense approach to inhibittranslation of endogenous follistatin-3 mRNA. Oligonucleotidescomplementary to the 5′ untranslated region of the mRNA should includethe complement of the AUG start codon. Antisense oligonucleotidescomplementary to mRNA coding regions are less efficient inhibitors oftranslation but could be used in accordance with the invention. Whetherdesigned to hybridize to the 5′-, 3′- or coding region of follistatin-3mRNA, antisense nucleic acids should be at least six nucleotides inlength, and are preferably oligonucleotides ranging from 6 to about 50nucleotides in length. In specific aspects the oligonucleotide is atleast 10 nucleotides, at least 17 nucleotides, at least 25 nucleotidesor at least 50 nucleotides.

[0473] The polynucleotides of the invention can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al.,1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810,published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988,BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988,Pharm. Res. 5:539-549). To this end, the oligonucleotide may beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, hybridization-triggered cleavageagent, etc.

[0474] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0475] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0476] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0477] In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

[0478] Polynucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0479] While antisense nucleotides complementary to the follistatin-3coding region sequence could be used, those complementary to thetranscribed untranslated region are most preferred.

[0480] Potential antagonists according to the invention also includecatalytic RNA, or a ribozyme (See, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy follistatin-3 mRNAs, the use ofhammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of follistatin-3 (FIGS. 1A, 1B, and 1C).Preferably, the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the follistatin-3 mRNA; i.e., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts.

[0481] As in the antisense approach, the ribozymes of the invention canbe composed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which expressfollistatin-3 in vivo. DNA constructs encoding the ribozyme may beintroduced into the cell in the same manner as described above for theintroduction of antisense encoding DNA. A preferred method of deliveryinvolves using a DNA construct “encoding” the ribozyme under the controlof a strong constitutive promoter, such as, for example, pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous follistatin-3 messagesand inhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

[0482] Antagonist/agonist compounds may be employed to inhibit the cellgrowth and proliferation effects of the polypeptides of the presentinvention on neoplastic cells and tissues, i.e. stimulation ofangiogenesis of tumors, and, therefore, retard or prevent abnormalcellular growth and proliferation, for example, in tumor formation orgrowth.

[0483] The antagonist/agonist may also be employed to preventhyper-vascular diseases, and prevent the proliferation of epitheliallens cells after extracapsular cataract surgery. Prevention of themitogenic activity of the polypeptides of the present invention may alsobe desirous in cases such as restenosis after balloon angioplasty.

[0484] The antagonist/agonist may also be employed to prevent the growthof scar tissue during wound healing.

[0485] The antagonist/agonist may also be employed to treat the diseasesdescribed herein.

[0486] Other Activities.

[0487] The polypeptide of the present invention, as a result of theability to stimulate vascular endothelial cell growth, may be employedin treatment for stimulating re-vascularization of ischemic tissues dueto various disease conditions such as thrombosis, arteriosclerosis, andother cardiovascular conditions. These polypeptide may also be employedto stimulate angiogenesis and limb regeneration, as discussed above.

[0488] The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

[0489] The polypeptide of the present invention may also be employedstimulate neuronal growth and to treat and prevent neuronal damage whichoccurs in certain neuronal disorders or neuro-degenerative conditionssuch as Alzheimer's disease, Parkinson's disease, and AIDS-relatedcomplex. Follistatin-3 may have the ability to stimulate chondrocytegrowth, therefore, they may be employed to enhance bone and periodontalregeneration and aid in tissue transplants or bone grafts.

[0490] The polypeptide of the present invention may be also be employedto prevent skin aging due to sunburn by stimulating keratinocyte growth.

[0491] The follistatin-3 polypeptide may also be employed for preventinghair loss, Since FGF family members activate hair-forming cells andpromotes melanocyte growth. Along the same lines, the polypeptides ofthe present invention may be employed to stimulate growth anddifferentiation of hematopoietic cells and bone marrow cells when usedin combination with other cytokines.

[0492] The follistatin-3 polypeptide may also be employed to maintainorgans before transplantation or for supporting cell culture of primarytissues.

[0493] The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

[0494] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, may also increase or decrease thedifferentiation or proliferation of embryonic stem cells, besides, asdiscussed above, hematopoietic lineage.

[0495] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, follistatin-3 polynucleotides orpolypeptides, or agonists or antagonists of follistatin-3, may be usedto modulate mammalian metabolism affecting catabolism, anabolism,processing, utilization, and storage of energy.

[0496] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, may be used to change a mammal's mentalstate or physical state by influencing biorhythms, caricadic rhythms,depression (including depressive disorders), tendency for violence,tolerance for pain, reproductive capabilities (preferably by Activin orInhibin-like activity), hormonal or endocrine levels, appetite, libido,memory, stress, or other cognitive qualities.

[0497] Follistatin-3 polynucleotides or polypeptides, or agonists orantagonists of follistatin-3, may also be used as a food additive orpreservative, such as to increase or decrease storage capabilities, fatcontent, lipid, protein, carbohydrate, vitamins, minerals, cofactors orother nutritional components.

[0498] The above-recited applications have uses in a wide variety ofhosts. Such hosts include, but are not limited to, human, murine,rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig,micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, andhuman. In specific embodiments, the host is a mouse, rabbit, goat,guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferredembodiments, the host is a mammal. In most preferred embodiments, thehost is a human.

[0499] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

[0500] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

[0501] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1(a) Expression and Purification of “His-Tagged”Follistatin-3 in E. coli.

[0502] The bacterial expression vector pHE-4 is used for bacterialexpression in this example. pHE-4 encodes ampicillin antibioticresistance (“Ampr”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”),six codons encoding histidine residues that allow affinity purificationusing trilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that an inserted DNA fragment encodinga polypeptide expresses that polypeptide with the six His residues(i.e., a “6 X His tag”) covalently linked to the amino terminus of thatpolypeptide.

[0503] The DNA sequence encoding the desired portion of thefollistatin-3 protein comprising the mature form of the follistatin-3amino acid sequence is amplified from the deposited cDNA clone using PCRoligonucleotide primers which anneal to the amino terminal sequences ofthe desired portion of the follistatin-3 protein and to sequences in thedeposited construct 3′ to the cDNA coding sequence. Additionalnucleotides containing restriction sites to facilitate cloning in thepHE-4 vector are added to the 5′ and 3′ primer sequences, respectively.

[0504] For cloning the mature form of the follistatin-3 protein, the 5′primer has the sequence 5′ TCA CGC CAT ATG GGC TCG GGG AAC C 3′ (SEQ IDNO:12) containing the underlined Nde I restriction site followed by 16nucleotides of the amino terminal coding sequence of the maturefollistatin-3 sequence in SEQ ID NO:2. One of ordinary skill in the artwould appreciate, of course, that the point in the protein codingsequence where the 5′ primer begins may be varied to amplify a DNAsegment encoding any desired portion of the complete follistatin-3protein shorter or longer than the mature form of the protein. The 3′primer has the sequence 5′CAT CCG GGT ACC TTA TTA CAC GAA GTT CTC TTCCTC TTC TG 3′ (SEQ ID NO:13) containing the underlined Asp 718restriction site followed by two stop codons and 23 nucleotidescomplementary to the 3′ end of the coding sequence of the follistatin-3DNA sequence in FIG. 1A.

[0505] The amplified follistatin-3 DNA fragment and the vector pHE4 aredigested with Nde I and Asp 718 and the digested DNAs are then ligatedtogether. Insertion of the follistatin-3 DNA into the restricted pHE4vector places the follistatin-3 protein coding region downstream fromthe IPTG-inducible promoter and in-frame with an initiating AUG and thesix histidine codons.

[0506] The ligation mixture is transformed into competent E. coli cellsusing standard procedures such as those described by Sambrook andcolleagues (Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). E. colistrain M15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance (“Kanr”),is used in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressingfollistatin-3 protein, is available commercially (QIAGEN, Inc., supra).Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

[0507] Clones containing the desired constructs are grown overnight(“O/N”) in liquid culture in LB media supplemented with both ampicillin(100 μg/ml) and kanamycin (25 μg/ml). The O/N culture is used toinoculate a large culture, at a dilution of approximately 1:25 to 1:250.The cells are grown to an optical density at 600 nm (“OD600”) of between0.4 and 0.6. Isopropyl-beta-D-thiogalactopyranoside (“IPTG”) is thenadded to a final concentration of 1 mM to induce transcription from thelac repressor sensitive promoter, by inactivating the lacI repressor.Cells subsequently are incubated further for 3 to 4 hours. Cells thenare harvested by centrifugation.

[0508] The cells are then stirred for 3-4 hours at 4 C in 6Mguanidine-HCl, pH 8. The cell debris is removed by centrifugation, andthe supernatant containing the follistatin-3 is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (QIAGEN,Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA resin withhigh affinity and can be purified in a simple one-step procedure (fordetails see: The QIAexpressionist, 1995, QIAGEN, Inc., supra). Brieflythe supernatant is loaded onto the column in 6 M guanidine-HCl pH 8, thecolumn is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, thenwashed with 10 volumes of 6 M guanidine-HCl pH 6, and finally thefollistatin-3 is eluted with 6 M guanidine-HCl, pH 5.

[0509] The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

[0510] The following alternative method may be used to purifyfollistatin-3 expressed in E coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10 C.

[0511] Upon completion of the production phase of the E. colifermentation, the cell culture is cooled to 4-10 C and the cells areharvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech).On the basis of the expected yield of protein per unit weight of cellpaste and the amount of purified protein required, an appropriate amountof cell paste, by weight, is suspended in a buffer solution containing100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to ahomogeneous suspension using a high shear mixer.

[0512] The cells ware then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

[0513] The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and thefollistatin-3 polypeptide-containing supernatant is incubated at 4 Covernight to allow further GuHCl extraction.

[0514] Following high speed centrifugation (30,000×g) to removeinsoluble particles, the GuHCl solubilized protein is refolded byquickly mixing the GuHCl extract with 20 volumes of buffer containing 50mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. Therefolded diluted protein solution is kept at 4 C without mixing for 12hours prior to further purification steps.

[0515] To clarify the refolded follistatin-3 polypeptide solution, apreviously prepared tangential filtration unit equipped with 0.16micrometer membrane filter with appropriate surface area (e.g.,Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed.The filtered sample is loaded onto a cation exchange resin (e.g., PorosHS-50, Perseptive Biosystems). The column is washed with 40 mM sodiumacetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mMNaCl in the same buffer, in a stepwise manner. The absorbance at 280 mmof the effluent is continuously monitored. Fractions are collected andfurther analyzed by SDS-PAGE.

[0516] Fractions containing the follistatin-3 polypeptide are thenpooled and mixed with 4 volumes of water. The diluted sample is thenloaded onto a previously prepared set of tandem columns of strong anion(Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20,Perseptive Biosystems) exchange resins. The columns are equilibratedwith 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mMsodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then elutedusing a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mMsodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5.Fractions are collected under constant A₂₈₀ monitoring of the effluent.Fractions containing the follistatin-3 polypeptide (determined, forinstance, by 16% SDS-PAGE) are then pooled.

[0517] The resultant follistatin-3 polypeptide exhibits greater than 95%purity after the above refolding and purification steps. No majorcontaminant bands are observed from Commassie blue stained 16% SDS-PAGEgel when 5 micrograms of purified protein is loaded. The purifiedprotein is also tested for endotoxin/LPS contamination, and typicallythe LPS content is less than 0.1 ng/ml according to LAL assays.

Example 2 Cloning and Expression of Follistatin-3 Protein in aBaculovirus Expression System.

[0518] In this illustrative example, the plasmid shuttle vector pA2 isused to insert the cloned DNA encoding complete protein, including itsnaturally associated secretory signal (leader) sequence, into abaculovirus to express the mature follistatin-3 protein, using standardmethods as described by Summers and colleagues (A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555 (1987)). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as Bam HI, Xba I and Asp 718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned polynucleotide.

[0519] Many other baculovirus vectors could be used in place of thevector above, such as pAc373, pVL941 and pAcIM1, as one skilled in theart would readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, by Luckow andcoworkers (Virology 170:31-39 (1989)).

[0520] The cDNA sequence encoding the full length follistatin-3 proteinin the deposited clone, including the AUG initiation codon and thenaturally associated leader sequence shown in SEQ ID NO:2, is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the gene. The 5′ primer has the sequence 5′CAT CGC GGA TCCGCC ATC ATG CGT CCC GGG GCG CCA GGG C 3′ (SEQ ID NO:14) containing theunderlined Bam HI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.196:947-950 (1987)), followed by 22 of nucleotides of the sequence ofthe complete follistatin-3 protein shown in FIG. 1A, beginning with theAUG initiation codon. The 3′ primer has the sequence 5′CAT CCG GGT ACCTCA CAC GAA GTT CTC TTC CTC TTC TG 3′ (SEQ ID NO:15) containing theunderlined Asp 718 restriction site followed by 23 nucleotidescomplementary to the 3′ noncoding sequence in FIG. 1A.

[0521] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with Bam HI and Asp 718 and againis purified on a 1% agarose gel. This fragment is designated herein F1.

[0522] The plasmid is digested with the restriction enzymes Bam HI andAsp 718 and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1”.

[0523] Fragment F1 and the dephosphorylated plasmid VI are ligatedtogether with T4 DNA ligase. E. coli HB101 or other suitable E. colihosts such as XL-1 Blue (Statagene Cloning Systems, La Jolla, Calif.)cells are transformed with the ligation mixture and spread on cultureplates. Bacteria are identified that contain the plasmid with the humanfollistatin-3 gene by digesting DNA from individual colonies using BamHI and Asp 718 and then analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing. This plasmid is designated herein pA2Follistatin-3.

[0524] Five μg of the plasmid pA2Follistatin-3 is co-transfected with1.0 μg of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner and colleaguew (Proc. Natl.Acad. Sci. USA 84:7413-7417 (1987)). One μg of BaculoGold™ virus DNA and5 μg of the plasmid pA2Follistatin-3 are mixed in a sterile well of amicrotiter plate containing 50 μl of serum-free Grace's medium (LifeTechnologies Inc., Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus90 μl Grace's medium are added, mixed and incubated for 15 minutes atroom temperature. Then the transfection mixture is added drop-wise toSf9 insect cells (ATCC® CRL 1711) seeded in a 35 mm tissue culture platewith 1 ml Grace's medium without serum. The plate is then incubated for5 hours at 27° C. The transfection solution is then removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum is added. Cultivation is then continued at 27° C. for four days.

[0525] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith (supra). An agarose gelwith “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10). After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-Follistatin-3.

[0526] To verify the expression of the follistatin-3 gene Sf9 cells aregrown in Grace's medium supplemented with 10% heat-inactivated FBS. Thecells are infected with the recombinant baculovirus V-Follistatin-3 at amultiplicity of infection (“MOI”) of about 2. If radiolabeled proteinsare desired, 6 hours later the medium is removed and is replaced withSF900 II medium minus methionine and cysteine (available from LifeTechnologies Inc., Rockville, Md.). After 42 hours, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

[0527] Microsequencing of the amino acid sequence of the amino terminusof purified protein may be used to determine the amino terminal sequenceof the mature form of the follistatin-3 protein, and thus the cleavagepoint and length of the naturally associated secretory signal peptide.

Example 3 Cloning and Expression of Follistatin-3 in Mammalian Cells

[0528] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of mRNA, theprotein coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC® 37152), pSV2dhfr (ATCC®37146) and pBC12MI (ATCC® 67109). Mammalian host cells that could beused include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 andC127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

[0529] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, hygromycin allowsthe identification and isolation of the transfected cells.

[0530] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful to develop cell lines that carry several hundred oreven several thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutamine synthase (GS; Murphy, et al.,Biochem J. 227:277-279 (1991); Bebbington, et al., Bio/Technology10:169-175 (1992)). Using these markers, the mammalian cells are grownin selective medium and the cells with the highest resistance areselected. These cell lines contain the amplified gene(s) integrated intoa chromosome. Chinese hamster ovary (CHO) and NSO cells are often usedfor the production of proteins.

[0531] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen, et al., Mol. Cel. Biol.5:438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart, et al.,Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites Bam HI, Xba I and Asp 718, facilitatethe cloning of the gene of interest. The vectors contain in addition the3′ intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

[0532] The expression plasmid, pFollistatin-3HA, is made by cloning aportion of the cDNA encoding the mature form of the follistatin-3protein into the expression vector peDNAI/Amp or pcDNAIII (which can beobtained from Invitrogen, Inc.).

[0533] The expression vector pcDNAI/amp contains: (1) an E. coli originof replication effective for propagation in E. coli and otherprokaryotic cells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson and colleagues (Cell 37:767 (1984)). The fusion of the HA tag tothe target protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

[0534] A DNA fragment encoding the complete follistatin-3 polypeptide iscloned into the polylinker region of the vector so that recombinantprotein expression is directed by the CMV promoter. The plasmidconstruction strategy is as follows. The follistatin-3 cDNA of thedeposited clone is amplified using primers that contain convenientrestriction sites, much as described above for construction of vectorsfor expression of follistatin-3 in E. coli. Suitable primers include thefollowing, which are used in this example. The 5′ primer, containing theunderlined Bam HI site, a Kozak sequence, an AUG start codon, and 22nucleotides of the 5′ coding region of the complete follistatin-3polypeptide, has the following sequence: 5′-CAT CGC GGA TCC GCC ACC ATGCGT CCC GGG GCG CCA GGG C-3′ (SEQ ID NO:16). The 3′ primer, containingthe underlined Asp 718 and 23 of nucleotides complementary to the 3′coding sequence immediately before the stop codon, has the followingsequence: 5′-TCA CCG CTC GAG CAC GAA GTT CTC TTC CTC TTC TG-3′ (SEQ IDNO:17).

[0535] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, aredigested with Bam HI and Asp 718 and then ligated. The ligation mixtureis transformed into E. coli strain SURE (Stratagene Cloning Systems, LaJolla, Calif. 92037), and the transformed culture is plated onampicillin media plates which then are incubated to allow growth ofampicillin resistant colonies. Plasmid DNA is isolated from resistantcolonies and examined by restriction analysis or other means for thepresence of the fragment encoding the complete follistatin-3 polypeptide

[0536] For expression of recombinant follistatin-3, COS cells aretransfected with an expression vector, as described above, usingDEAE-dextran, as described, for instance, by Sambrook and coworkers(Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press,Cold Spring Harbor, N.Y. (1989)). Cells are incubated under conditionsfor expression of follistatin-3 by the vector.

[0537] Expression of the follistatin-3-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow and colleagues (Antibodies: A Laboratory Manual, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)).To this end, two days after transfection, the cells are labeled byincubation in media containing ³⁵S-cysteine for 8 hours. The cells andthe media are collected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson andcolleagues (supra). Proteins are precipitated from the cell lysate andfrom the culture media using an HA-specific monoclonal antibody. Theprecipitated proteins then are analyzed by SDS-PAGE and autoradiography.An expression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b) Cloning and Expression in CHO Cells

[0538] The vector pC4 is used for the expression of follistatin-3polypeptide. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr(ATCC®) Accession No. 37146). The plasmid contains the mouse DHFR geneunder control of the SV40 early promoter. Chinese hamster ovary- orother cells lacking dihydrofolate activity that are transfected withthese plasmids can be selected by growing the cells in a selectivemedium (alpha minus MEM, Life Technologies) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented (see,e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin,J. L. and Ma, C. Biochem. et Biophys. Acta, 1097:107-143 (1990); Page,M. J. and Sydenham, M. A. Biotechnology 9:64-68 (1991)). Cells grown inincreasing concentrations of MTX develop resistance to the drug byoverproducing the target enzyme, DHFR, as a result of amplification ofthe DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach may be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained which contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

[0539] Plasmid pC4 contains for expressing the gene of interest thestrong promoter of the long terminal repeat (LTR) of the Rouse SarcomaVirus (Cullen, et al., Mol. Cell. Biol. 5:438-447 (1985)) plus afragment isolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV; Boshart, et al., Cell 41:521-530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: Bam HI, Xba I,and Asp 718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the follistatin-3 polypeptide in aregulated way in mammalian cells (Gossen, M., and Bujard, H. Proc. Natl.Acad. Sci. USA 89:5547-5551 (1992)). For the polyadenylation of the mRNAother signals, e.g., from the human growth hormone or globin genes canbe used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

[0540] The plasmid pC4 is digested with the restriction enzymes Bam HIand Asp 718 and then dephosphorylated using calf intestinal phosphatesby procedures known in the art. The vector is then isolated from a 1%agarose gel.

[0541] The DNA sequence encoding the complete follistatin-3 polypeptideis amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the desired portion of the gene. The 5′ primercontaining the underlined Bam HI site, a Kozak sequence, an AUG startcodon, and 22 nucleotides of the 5′ coding region of the completefollistatin-3 polypeptide, has the following sequence: 5′CAT CGC GGA TCCGCC ACC ATG CGT CCC GGG GCG CCA GGG C 3′ (SEQ ID NO:18). The 3′ primer,containing the underlined Asp 718 restriction site and 23 of nucleotidescomplementary to the 3′ coding sequence immediately before the stopcodon as shown in FIG. 1A (SEQ ID NO:1), has the following sequence:5′CAT CCG GGT ACC TCA CAC GAA GTT CTC TTC CTC TTC TG 3′ (SEQ ID NO:19).

[0542] The amplified fragment is digested with the endonucleases Bam HIand Asp 718 and then purified again on a 1% agarose gel. The isolatedfragment and the dephosphorylated vector are then ligated with T4 DNAligase. E. coli HB101 or XL-1 Blue cells are then transformed andbacteria are identified that contain the fragment inserted into plasmidpC4 using, for instance, restriction enzyme analysis.

[0543] Chinese hamster ovary cells lacking an active DHFR gene are usedfor transfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner, et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G41 8. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 4 Tissue Distribution of Follistatin-3 mRNA Expression

[0544] Northern blot analysis was carried out to examine follistatin-3gene expression in human tissues, using methods described by, amongothers, Sambrook and colleagues (supra). A cDNA probe containing theentire nucleotide sequence of the follistatin-3 protein (SEQ ID NO:1)was labeled with ³²p using the rediprimetm DNA labeling system (AmershamLife Science), according to manufacturer's instructions. After labeling,the probe was purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe was then used to examine varioushuman tissues for follistatin-3 mRNA.

[0545] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) were obtained fromClontech and were examined with the labeled probe using ExpressHyb™hybridization solution (Clontech) according to manufacturer's protocolnumber PT 1190-1. Following hybridization and washing, the blots weremounted and exposed to film at −70° C. overnight, and films developedaccording to standard procedures. The follistatin-3-specific proberecognized an mRNA species of approximately 2.6 kb in most tissuesexamined.

Example 5 Follistatin-3 Encodes an Activin-Bin Ding Protein that isDifferentially Regulated Compared to Follistatin.

[0546] Follistatin is a glycoslylated, single chain polypeptide that wasdiscovered as an activin-binding protein in the rat ovary. (Vale, W., etal., Nature 321, 776-79 (1986); Ling, N., et al., Nature 321, 779-82(1986); Ueno, N., et al., Proc. Natl. Acad. Sci. U.S.A. 84, 8282-86(1987); Esch, F. S., et al., Mol. Endocrinol. 1, 849-55 (1987);Nakamura, T., et al., Science 247, 836-38 (1990), reviewed in Michel,V., et al., Molec. Cell. Endocrinol. 91, 1-11 (1993)). It was shown toinhibit the release of follicle-stimulating hormone (FSH) from thepituitary by binding to activins (Nakamura, T., et al., Science 247,836-38 (1990); Kogawa, K., et al., Endocrinol. 128, 1434-40 (1991),reviewed in Vale, W., et al., The inhibin/activin family of hormones andgrowth factors, In Peptide Growth Factors and Their Receptors II, eds.Spom, M. B. and Roberts, A. B., (Springer-Verlag) (1990)), indicatingthat follistatin inhibits activin actions. This has now been confirmedin in vitro and in vivo experiments (Nakamura, T., et al., Science 247,836-38 (1990); de Winter, J. P., et al., Mol. Cell. Endocrinol. 116,105-14 (1996); for review see Patel, K. Int. J. Biochem. Cell Biol. 30,1087-93 (1998)). Follistatin prevents binding of activins to their highaffinity transmembrane receptors (de Winter, J. P., et al., Mol. Cell.Endocrinol. 116, 105-14 (1996)) and it also facilitates clearance ofactivins by accelerating their endocytotic degradation (Hashimoto, O.,et al., J. Biol. Chem. 272, 13835-13842 (1997)).

[0547] Two forms of follistatin have been identified, follistatin-315(FS-315) and a carboxyterminally truncated variant, follistatin-288(FS-288). These two follistatin molecules result from alternativesplicing of one primary transcript (Shimasaki, S., et al., Proc., Natl.,Acad. Sci. U.S.A. 85, 4218-22 (1998)). Among several differences, FS-288was shown to be more potent than FS-315 in suppressing FSH release inrat anterior pituitary cells (Sugino, K., et al., J. Biol. Chem. 268,15579-87 (1993)). In addition to its activin-binding activity,follistatin binds to other members of the transforming growth factor(TGF)-beta superfamily such as bone morphogenetic proteins 2, 4, and 7(Fainsod. A., et al., Mech. Dev. 63, 39-50 (1997)). The in vivorelevance of these interactions is as yet unclear. The physiologicalrole of follistatin is not limited to the inhibition of FSH release. Forexample, overexpression of follistatin in the developing Xenopus laevisembryo led to induction of neural tissue (Hemmati-Brivanlou, A., et al.,Cell 77, 283-95 (1994)). Furthermore, follistatin-deficient mice showedmultiple defects and perinatal death (Matzuk, M. M., et al., Nature 374,360-63 (1995)). The defects include those in the muscles, skin, bone andteeth. These data are consistant with the hypothesis that follistatin isinvolved in multiple physiological functions.

[0548] We have recently identified a human follistatin homologue,follistatin-3, from the Human Genome Sciences expressed sequence tag(EST) database (EST clone number HDTAH85). The cDNA of this follistatinhomologue is 2.6 kb in length and encodes a protein of 263 amino acids.Hayette et al. have also reported the Follistatin-Related Gene (FLRG)(Hayette, S., et al., Oncogene 16, 2949-54 (1998)) whose sequence wasidentical to our cDNA. FLRG was identified by positional cloning oftranscriptional units on chromosome 19 at the t(11; 19)(q13; p13)translocation breakpoint. This breakpoint was observed in a case of Bcell chronic lymphocytic leukemia. Hayette et al. have also reported onthe structural rearrangement of the FLRG locus in a case ofnon-Hodgkin's lymphoma. These data suggest that FLRG may play a role inleukemogenesis.

[0549] In this study, we demonstrate that FLRG is a functionalactivin-binding protein which, like follistatin, binds both activin Aand activin B. However, we demonstrate differential expression intissues and regulation of follistatin and FLRG expression in culturedkeratinocytes. Our results indicate differences in the in vivoregulation and functions of FLRG and follistatin proteins.

[0550] Materials and Methods

[0551] Molecular Cloning of Follistatin-3. Searches of the Human GenomeSciences database of expressed sequence tags (ESTs) identified a cDNAclone from a Hodgkin's Lymphoma II library, HDTAH85, that was homologousto part of follistatin. We named this clone follistatin-3. A cDNA probeencompassing the first 417 nucleotides of the predicted coding sequenceof follistatin-3 was isolated by performing two rounds of PCR from ahuman adult liver cDNA library. This FLRG-specific probe was used toobtain a longer clone containing the complete coding sequence and the 3′non-coding region of follistatin-3 from a fetal bone cDNA library.

[0552] Transfection and immunoprecipitation experiments. Fortransfection into mammalian cells, the FLRG open reading frame without5′- or 3′-untranslated regions was cloned into pcDNA3 vector(Invitrogen) with an in-frame epitope tag, HA (YPYDVPDYA) (SEQ IDNO:20), or Flag (DYKDDDDK) (SEQ ID NO:21) at the carboxyl terminus.Likewise, the human activin betaA open reading frame without 5′- or3′-untranslated regions was cloned into pcDNA3 vector (Invitrogen) withan in-frame HA tag, and the human activin betaB with an in-frame HA tagor a myc tag (EQKLISEEDL) (SEQ ID NO:22), at the carboxyl termini. Humankidney epithelial 293 cells (ATCC®), grown in Dulbecco's modified Eaglesmedium (DMEM) supplemented with 10% fetal calf serum (FCS), weretransfected with expression constructs, such as pcDNA-FLRG-HA,pcDNA-FLRG-Flag, or combinations of FLRG constructs with activin betaAor activin betaB constructs. The transfection was performed using theLipofectamine method (Life Technology) according to the manufacturer'sinstructions. The cell culture media were collected 48 hourspost-transfection. Cells were harvested at the same time as the culturemedia. Transfected c ells were lysed in NP-40 lysis buffer (20 mM Tris,pH 8.0, 137 mM NaCl, 1% NP-40, 10% Glycerol, 1 mM PMSF, Leupeptin 1.0μg/ml, EDTA 0.5 mM, NaF 1 mM, E64 1 μg/ml, Aprorinin 1 μg/ml sodiumorthovanadate 1 mM,). FLRG proteins in the cell culture supernatants andcell lysates were immunoprecipitated with a monoclonal anti-HA antibody(Roche, Mannheim, Germany; 2 micrograms/10 ml for cell supernatants and1 microgram/ml for cell lysates), or a monoclonal anti-Flag antibody(Kodak-IBI). The immunoprecipitates were separated by reducing SDS PAGEand subsequently by Western blot analyses.

[0553] Anti-FLRG antibody. Purified FLRG protein fromBaculoviraus/insect cell expression system was used to immunize rabbits.The anti-serum was used at 1:7,500 dilution for Western blotting.

[0554] Northern blot analysis. Three Clontech human multiple tissueNorthern blots were hybridized with ³²P-labelled FLRG and follistatincDNA probes as described by the manufacturer.

[0555] Cell culture for RNase protection assays. The immortalized butnon-transformed human HaCaT keratinocyte cell line (18) was used for alltissue culture experiments. Cells were cultured in Dulbecco's modifiedEagles medium (DMEM) supplemented with 10% fetal calf serum. For theanalysis of follistatin and FLRG regulation, cells were grown toconfluency in 10-cm culture dishes and rendered quiescent by a 16 hrincubation in serum-free DMEM. Cells were subsequently incubated forvarying periods in fresh DMEM containing 20 ng/ml epidermal growthfactor (EGF), 10 ng/ml keratinocyte growth factor (KGF), or 1 ng/mltransforming growth factor betal (TGF-betal). Aliquots of cells wereharvested before and at different time points after treatment with thesereagents and used for RNA isolation. FCS and DMEM were purchased fromLife Technologies, Inc. Growth factors and cytokines were from Roche(Mannheim, Germany). Each experiment was repeated at least twice.

[0556] RNA isolation and RNase protection assay. Total cellular RNA wasisolated from HaCaT cells as described by Chomczynski and Sacchi(Chomczynski, P. and Sacchi, N. Anal. Biochem. 162, 156-59 (1987)).RNase protection assays were carried out as described (Werner, S., etal., EMBO J. 12, 2635-43 (1993)). All protection assays were repeatedwith a different set of RNAs from independent experiments. A 247 bpfragment corresponding to nt 28-274 of the human FLRG cDNA and a 253 bpfragment corresponding to nt 28-280 of the folliststin gene (Shimasaki,S., et al., Proc., Natl., Acad. Sci. U.S.A. 85, 4218-22 (1998)) wereused as probes.

[0557] Results and Discussion

[0558] Expression of FLRG in Mammalian Cells

[0559] To elucidate the functions of FLRG, we first expressed FLRGprotein in mammalian cells. An expression vector containing the entireopen reading frame of FLRG fused to a C-terminal epitope tag, Flag, wastransfected into 293 human kidney epithelial cell. Cell lysates and cellculture media were analyzed by Western blot analyses. In both celllysates and cell culture media, FLRG appeared as a diffuse band ofapproximately 35 kDa on a Western blot probed with an anti-FLRGantibody. The same 35 kDa band was detected with an anti-tag antibodythat recognized the C-terminal Flag-tag on FLRG. The amino acid sequenceof FLRG contains a signal peptide and therefore predicts that FLRG is asecreted protein. Our results showed that the FLRG protein was indeedsecreted into the cell culture media. However, unlike the intracellularFLRG protein, the secreted FLRG detected by the anti-Flag antibodyshowed a much weaker signal than that detected by the polyclonalanti-FLRG antibody. One hypothesis for this observation could be thatpart of the secreted FLRG may have a proteolytically clipped C-terminus.The predicted molecular weight of FLRG is 27.6 kDa. The differencebetween the predicted and the apparent molecular weight (35 kDa) couldbe accounted for by glycosylation of the FLRG protein. In fact, the FLRGamino acid sequence contains two predicted N-glycosylation sites (aa 73and aa 215) (Hayette, S., et al., Oncogene 16, 2949-54 (1998)).Furthermore, thue diffuseness of the FLRG protein band on the Westernblot also suggests that FLRG is glycosylated.

[0560] Binding of FLRG to activin A and B

[0561] Activin exists as homo- or heterodimers of a betaA and a betaBchain. Three activins, activin A (betaAbetaA homodimer), activin B(betaBbetaB homodimer), and activin AB (betaAbetaB heterodimer) all bindfollistatin with high affinities. Activins are synthesized as largeprecursor polypeptides which are proteolytically processed into maturepolypeptides. The disulfide-linked dimers of mature activins form thebiologically active molecules of approximately 28 kDa. Under reducingconditions, the dimers can be separated into two subunits ofapproximately 14 kDa (reviewed in Vale, W., et al., The inhibin/activinfamily of hormones and growth factors, In Peptide Growth Factors andTheir Receptors II, eds. Sporn, M. B. and Roberts, A. B.,(Springer-Verlag) (1 990)).

[0562] To determine whether FLRG is able to bind activins, 293 cellswere co-transfected with a C-terminal Flag-tagged FLRG construct witheither an activin betaA-HA or with an activin betaB-myc expressionconstruct. Flag-tagged follistatin-315 (FS-315) and Flag-taggedfollistatin-288 (FS-288), were used as positive controls. Thesemolecules were immunoprecipitated from either the cell culture media orcell lysates with an anti-Flag monoclonal antibody. Theco-immunoprecipitated activins A or B were then detected on a Westernblot by anti-tag antibodies. Both positive controls, FS-315 and FS-288,co-immunoprecipitated the mature form of Flag-tagged activin A whichappeared on the Western blot as the 16 kDa monomer under reducingconditions. In addition, we made a novel observation that FS-315 andFS-288 immunoprecipitated a protein of approximately 55 kDa from thecell lysates. The latter most likely represents the reduced form ofactivin A precursor. Although this precursor was reported to be inactivein releasing follicle-stimulating hormone from pituitary cells in invitro assays (Mason, A. J., et al., Mol. Endocrinol. 10: 1055-65(1996)), follistatin binding to the precursor may prevent the processingof the precursor into the active mature form of activin A. This may, inturn, further inhibit activin A activity.

[0563] In cell lysates where FLRG was co-transfected with theactivin-betaA cDNA, FLRG co-immunoprecipitated activin A. The ability ofFLRG to associate with activin A was comparable to FS-315 and FS-288, asjudged from the amounts of FLRG, FS-315, and FS-288 proteins in theimmunoprecipitates. The major activin A species precipitated from celllysates by FLRG was the high molecular weight form of ˜55 kDa underreducing conditions. In control experiments where activin betaA wastransfected alone or co-transfected with pcDNA vector, no activin A wasimmunoprecipitated by the anti-Flag antibody that recognized FLRG. Theseresults demonstrate that FLRG, like FS-315 and FS-288, can bind theunprocessed high molecular weight activin A precursor.

[0564] In the cell culture media, secreted FLRG, as well as FS-315 andFS-288, co-immunoprecipitated the secreted low molecular weight matureform activin A, as demonstrated by the detection of the 16 kDa monomericFlag-tagged activin betaA protein on the Western blot. In addition tothe 16 kDa species, FS-288 also precipitated a secreted Flag-taggedactivin betaA species of approximately 20 kDa.

[0565] In cell lysates where FLRG was co-transfected with activin betaB,FLRG co-immunoprecipitated activin B precursor molecules of 48-55 kDa.Judging from the amounts of FLRG, FS-315, and FS-288 proteins in theimmunoprecipitates, the ability of FLRG to co-immunoprecipitate activinB was at least as good as that of FS-315 and possibly even better thanthat of FS-288. In control experiments in which activin betaB cDNA wastransfected alone or co-transfected with pcDNA vector, no activin B wasimmunoprecipitated by the anti-HA antibody that recognized FLRG.

[0566] In a reverse experiment where activin B was immunoprecipitated byan anti-myc antibody, FLRG, FS-315, and FS-288 all co-immunoprecipitatedwith activin B. These results further support the specificity of theinteraction between FLRG and activin B.

[0567] In the cell culture media, secreted FLRG co-immunoprecipitatedthe secreted, low molecular weight mature form of activin B asdemonstrated by the detection of the 14 kDa monomeric betaB protein onthe Western blot. In addition, secreted FLRG also co-immunoprecipitatedthe secreted high molecular weight precursor form of activin B asdemonstrated by the detection of the 55 kDa monomeric betaB protein onthe Western blot.

[0568] Differential expression of FLRG and follistatin.

[0569] To gain insight into possible in vivo functions of FLRG, weanalyzed FLRG and follistatin expression in various human tissues. FLRGwas expressed at highest levels in the testis, adrenal glands, lung,heart and liver. Lower levels of FLRG transcripts were seen in thestomach, small intestine, colon, pancreas, thyroid, ovary and prostate.FLRG mRNA could not be detected in the thymus, kidney, skeletal muscle,liver, brain, peripheral blood lymphocytes and spleen. The expressionpattern of follistatin mRNA revealed obvious similarities to that ofFLRG, although this molecule was also expressed in the liver. The FLRGexpression data extend the results obtained by Hayette et al. (Hayette,S., et al., Oncogene 16, 2949-54 (1998)), who found expression of thismolecule in murine heart, lung, testis, and kidney. In contrast to thesedata, however, we could not detect FLRG mRNA in the human kidney.

[0570] To determine potential differences in the regulation offollistatin and FLRG expression, we analyzed the effect of variousgrowth factors and cytokines on the expression of these genes in HaCaTkeratinocytes. This cell line was chosen since it expresses fairly highlevels of both follistatin and FLRG mRNA. Cells were rendered quiescentby serum starvation and treated with the epithelial mitogens KGF andEGF. Follistatin and FLRG mRNAs were hardly detectable in quiescentkeratinocytes. Upon addition of the growth factors, a strong inductionof follistatin expression was observed, whereby maximal levels were seen8 hours after addition of the mitogen. EGF was the more potent inducercompared to KGF. This induction was long-lasting and follistatin mRNAlevels had still not returned to basal levels 24 hours after growthfactor stimulation. In contrast to follistatin, FLRG induction occurredalready within one hour after addition of the growth factors. However,the degree of induction was significantly lower.

[0571] To determine whether follistatin and FLRG mRNA induction isspecific for epithelial cell mitogens, we analyzed the effect ofTGF-betal, a strong inhibitor of keratinocyte proliferation.Interestingly, TGF-betal caused a strong induction of both FS and FLRGexpression, whereby the degree of induction was similar for both genes.Similar as with EGF and KGF, induction of FLRG expression by TGF-betaloccurred earlier compared to follistatin.

[0572] Taken together, these results demonstrate differential regulationof follistatin and FLRG mRNA expression, indicating that the twoproteins might be available under different biological circumstances.The discovery of a novel activin-binding protein with a differentexpression pattern compared to follistatin necessitates thereinterpretation of various activin expression data, since the presenceof FLRG is likely to modulate the activity of the available activin.Whether this activin-FLRG interaction leads to inhibition of activinfunction as shown for follistatin remains to be elucidated.

Example 6 Production of an Antibody

[0573] a) Hybridoma Technology

[0574] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) As one exampleof such methods, cells expressing follistatin-3 is administered to ananimal to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of follistatin-3protein is prepared and purified to render it substantially free ofnatural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity.

[0575] In the most preferred method, the antibodies of the presentinvention are monoclonal antibodies (or protein binding fragmentsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology. (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur.J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976);Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures involveimmunizing an animal (preferably a mouse) with follistatin-3 polypeptideor, more preferably, with a secreted follistatin-3polypeptide-expressing cell. Such cells may be cultured in any suitabletissue culture medium; however, it is preferable to culture cells inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56 C), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100ug/ml of streptomycin.

[0576] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP2O), available fromthe ATCC®. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981).) Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding thefollistatin-3 polypeptide.

[0577] Alternatively, additional antibodies capable of binding tofollistatin-3 polypeptide can be produced in a two-step procedure usinganti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore, it is possible toobtain an antibody which binds to a second antibody. In accordance withthis method, protein specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to thefollistatin-3 protein-specific antibody can be blocked by follistatin-3.Such antibodies comprise anti-idiotypic antibodies to the follistatin-3protein-specific antibody and can be used to immunize an animal toinduce formation of further follistatin-3 protein-specific antibodies.

[0578] It will be appreciated that Fab and F(ab′)2 and other fragmentsof the antibodies of the present invention may be used according to themethods disclosed herein. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)2 fragments). Alternatively,secreted follistatin-3 protein-binding fragments can be produced throughthe application of recombinant DNA technology or through syntheticchemistry.

[0579] For in vivo use of antibodies in humans, it may be preferable touse “humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).)

[0580] b) Isolation of Antibody Fragments Directed Against Follistatin-3from a Library of scFvs.

[0581] Naturally occuring V-genes isolated from human PBLs areconstructed into a large library of antibody fragments which containreactivities against follistatin-3 to which the donor may or may nothave been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated hereinin its entirety by reference).

[0582] Rescue of the Library. A library of scFvs is constructed from theRNA of human PBLs as described in WO92/01047. To rescue phage displayingantibody fragments, approximately 109 E. coli harbouring the phagemidare used to inoculate 50 ml of 2xTY containing 1% glucose and 100 ug/mlof ampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking.Five ml of this culture is used to innoculate 50 ml of 2xTY-AMP-GLU,2×10⁸ TU of delta gene 3 helper (M13 delta gene III, see WO92/01047) areadded and the culture incubated at 37 C for 45 minutes without shakingand then at 37 C for 45 minutes with shaking. The culture is centrifugedat 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of of2xTY containing 100 ug/ml ampicillin and 50 ug/ml kanamycin and grownovernight. Phage are prepared as described in WO92/01047.

[0583] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious Ml 3 delta gene III particles are made by growingthe helper phage in cells harbouring a pUC19 derivative supplying thewild type gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37 C without shaking and then for a further hourat 37 C with shaking. Cells are spun down (IEC-Centra 8, 4000 revs/minfor 10 min), resuspended in 300 ml 2xTY broth containing 100 ugampicillin/ml and 25 ug kanamycin/ml (2xTY-AMP-KAN) and grown overnight,shaking at 37 C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 um filter (MinisartNML; Sartorius) to give a final concentration of approximately 10¹³transducing units/ml (ampicillin-resistant clones).

[0584] Panning of the Library. Immunotubes (Nunc) are coated overnightin PBS with 4 ml of either 100 ug/ml or 10 ug/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37 C and then washed 3 times in PBS. Approximately 10¹³ TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 11.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37 degreeC. The E. coli are then plated on TYE plates containing 1% glucose and100 ug/ml ampicillin. The resulting bacterial library is then rescuedwith delta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[0585] Characterization of Binders. Eluted phage from the 3rd and 4throunds of selection are used to infect E. coli HB 2151 and soluble scfvis produced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (seee.g., WO92/01047) and then by sequencing.

Example 7 Method of Determining Alterations in the Follistatin-3 Gene

[0586] RNA isolated from entire families or individual patientspresenting with a phenotype of interest (such as a disease) is beisolated. cDNA is then generated from these RNA samples using protocolsknown in the art. (See, Sambrook.) The cDNA is then used as a templatefor PCR, employing primers surrounding regions of interest in SEQ IDNO:1. Suggested PCR conditions consist of 35 cycles at 95 degree C. for30 seconds; 60-120 seconds at 52-58 degree C.; and 60-120 seconds at 70degree C., using buffer solutions described in Sidransky, D., et al.,Science 252:706 (1991).

[0587] PCR products are then sequenced using primers labeled at their 5′end with T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons offollistatin-3 is also determined and genomic PCR products analyzed toconfirm the results. PCR products harboring suspected mutations infollistatin-3 is then cloned and sequenced to validate the results ofthe direct sequencing.

[0588] PCR products of follistatin-3 are cloned into T-tailed vectors asdescribed in Holton, T. A. and Graham, M. W., Nucleic Acids Research,19:1156 (1991) and sequenced with T7 polymerase (United StatesBiochemical). Affected individuals are identified by mutations infollistatin-3 not present in unaffected individuals.

[0589] Genomic rearrangements are also observed as a method ofdetermining alterations in the follistatin-3 gene. Genomic clonesisolated according to methods well-known in the art are nick-translatedwith digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), andFISH performed as described in Johnson, Cg. et al., Methods Cell Biol.35:73-99 (1991). Hybridization with the labeled probe is carried outusing a vast excess of human cot-1 DNA for specific hybridization to thefollistatin-3 genomic locus.

[0590] Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of follistatin-3 (hybridized by theprobe) are identified as insertions, deletions, and translocations.These follistatin-3 alterations are used as a diagnostic marker for anassociated disease.

Example 8 Method of Detecting Abnormal Levels of Follistatin-3 in aBiological Sample

[0591] Follistatin-3 polypeptides can be detected in a biologicalsample, and if an increased or decreased level of follistatin-3 isdetected, this polypeptide is a marker for a particular phenotype.Methods of detection are numerous, and thus, it is understood that oneskilled in the art can modify the following assay to fit theirparticular needs.

[0592] For example, antibody-sandwich ELISAs are used to detectfollistatin-3 in a sample, preferably a biological sample. Wells of amicrotiter plate are coated with specific antibodies to follistatin-3,at a final concentration of 0.2 to 10 ug/ml. The antibodies are eithermonoclonal or polyclonal and are produced by the method described inExample 6. The wells are blocked so that non-specific binding offollistatin-3 to the well is reduced.

[0593] The coated wells are then incubated for >2 hours at RT with asample containing follistatin-3. Preferably, serial dilutions of thesample should be used to validate results. The plates are then washedthree times with deionized or distilled water to remove unboundedfollistatin-3.

[0594] Next, 50 ul of specific antibody-alkaline phosphatase conjugate,at a concentration of 25-400 ng, is added and incubated for 2 hours atroom temperature. The plates are again washed three times with deionizedor distilled water to remove unbounded conjugate.

[0595] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) orp-nitrophenyl phosphate (NPP) substrate solution to each well andincubate 1 hour at room temperature. Measure the reaction by amicrotiter plate reader. Prepare a standard curve, using serialdilutions of a control sample, and plot follistatin-3 polypeptideconcentration on the X-axis (log scale) and fluorescence or absorbanceof the Y-axis (linear scale). Interpolate the concentration of thefollistatin-3 in the sample using the standard curve.

Example 9 Formulation

[0596] The invention also provides methods of treatment and/orprevention of diseases, disorders, and/or conditions (such as, forexample, any one or more of the diseases, disorders, and/or conditionsdisclosed herein) by administration to a subject of an effective amountof a Therapeutic. By therapeutic is meant a polynucleotides orpolypeptides of the invention (including fragments and variants),agonists or antagonists thereof, and/or antibodies thereto, incombination with a pharmaceutically acceptable carrier type (e.g., asterile carrier).

[0597] The Therapeutic will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the Therapeutic alone), the site of delivery, the methodof administration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

[0598] As a general proposition, the total pharmaceutically effectiveamount of the Therapeutic administered parenterally per dose will be inthe range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the Therapeutic is typicallyadministered at a dose rate of about 1 ug/kg/hour to about 50ug/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

[0599] Therapeutics can be are administered orally, rectally,parenterally, intracistemally, intravaginally, intraperitoneally,topically (as by powders, ointments, gels, drops or transdermal patch),bucally, or as an oral or nasal spray. “Pharmaceutically acceptablecarrier” refers to a non-toxic solid, semisolid or liquid filler,diluent, encapsulating material or formulation auxiliary of any. Theterm “parenteral” as used herein refers to modes of administration whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

[0600] Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[0601] Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

[0602] Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

[0603] Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

[0604] In yet an additional embodiment, the Therapeutics of theinvention are delivered by way of a pump (see Langer, supra; Sefton, CRCCrit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507(1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

[0605] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0606] For parenteral administration, in one embodiment, the Therapeuticis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

[0607] Generally, the formulations are prepared by contacting theTherapeutic uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

[0608] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0609] The Therapeutic is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

[0610] Any pharmaceutical used for therapeutic administration can besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

[0611] Therapeutics ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 1 0-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

[0612] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the Therapeutics of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, theTherapeutics may be employed in conjunction with other therapeuticcompounds.

[0613] The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRLI005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0614] The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0615] In one embodiment, the Therapeutics of the invention areadministered in combination with members of the TNF family. TNF,TNF-related or TNF-like molecules that may be administered with theTherapeutics of the invention include, but are not limited to, solubleforms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AITM-I (International Publication No. WO97/33899), endokine-alpha (International Publication No. WO 98/07880),TR6 (International Publication No. WO 98/30694), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892),TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12,and soluble forms CD154, CD70, and CD153.

[0616] In certain embodiments, Therapeutics of the invention areadministered in combination with antiretroviral agents, nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat A/DS and/or to prevent ortreat HIV infection.

[0617] In other embodiments, Therapeutics of the invention may beadministered in combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONTAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™(filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE™, PENTAMIDINE, and/or ATOVAQUONE™to prophylactically treat or prevent an opportunistic Pneumocystiscarinii pneumonia infection. In another specific embodiment,Therapeutics of the invention are used in any combination withISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,Therapeutics of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

[0618] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

[0619] In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

[0620] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[0621] In specific embodiments, Therapeutics of the invention areadministered in combination with immunosuppressants. Immunosuppressantspreparations that may be administered with the Therapeutics of theinvention include, but are not limited to, ORTHOCLONE™ (OKT3),SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™ (tacrolimus),CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, andRAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants maybe used to prevent rejection of organ or bone marrow transplantation.

[0622] In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, Therapeutics of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

[0623] In an additional embodiment, the Therapeutics of the inventionare administered alone or in combination with an anti-inflammatoryagent. Anti-inflammatory agents that may be administered with theTherapeutics of the invention include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

[0624] In another embodiment, compostions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the Therapeuticsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0625] In a specific embodiment, Therapeutics of the invention areadministered in combination with CHOP (cyclophosphamide, doxorubicin,vincristine, and prednisone) or any combination of the components ofCHOP. In another embodiment, Therapeutics of the invention areadministered in combination with Rituximab. In a further embodiment,Therapeutics of the invention are administered with Rituxmab and CHOP,or Rituxmab and any combination of the components of CHOP.

[0626] In an additional embodiment, the Therapeutics of the inventionare administered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-1 8, IL-19, IL-20, and IL-21.

[0627] In an additional embodiment, the Therapeutics of the inventionare administered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-6821 10;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

[0628] In an additional embodiment, the Therapeutics of the inventionare administered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE™(SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

[0629] In an additional embodiment, the Therapeutics of the inventionare administered in combination with Fibroblast Growth Factors.Fibroblast Growth Factors that may be administered with the Therapeuticsof the invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

[0630] In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 10 Method of Treating Decreased Levels of Follistatin-3

[0631] The present invention relates to a method for treating anindividual in need of a decreased level of follistatin-3 activity in thebody comprising, administering to such an individual a compositioncomprising a therapeutically effective amount of follistatin-3antagonist. Preferred antagonists for use in the present invention arefollistatin-3-specific antibodies.

[0632] Moreover, it will be appreciated that conditions caused by adecrease in the standard or normal expression level of follistatin-3, inan individual can be treated by administering follistatin-3, preferablyin the secreted form. Thus, the invention also provides a method oftreatment of an individual in need of an increased level offollistatin-3 polypeptide comprising administering to such an individuala pharmaceutical composition comprising an amount of follistatin-3 toincrease the activity level of follstatin-3 in such an individual.

[0633] For example, a patient with decreased levels of follistatin-3polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide forsix consecutive days. Preferably, the polypeptide is in the secretedform. The exact details of the dosing scheme based on administration andformulation, are provided in Example 8.

Example 11 Method of Treating Increased Levels of Follistatin-3

[0634] The present invention also relates to a method for treating anindividual in need of an increased level of follistatin-3 activity inthe body comprising administering to such an individual a compositioncomprising a therapeutically effective amount of follistatin-3 or anagonist thereof.

[0635] Antisense technology is used to inhibit production offollistatin-3. This technology is one example of a method of decreasinglevels of follistatin-3 polypeptide, preferably a secreted form, due toa variety of etiologies, such as cancer.

[0636] For example, a patient diagnosed with abnormally increased levelsof follistatin-3 is administered intravenously antisense polynucleotidesat 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment isrepeated after a 7-day rest period if the treatment was well tolerated.The formulation of the antisense polynucleotide is provided in Example8.

Example 12 Method of Treatment Using Gene Therapy-Ex Vivo

[0637] One method of gene therapy transplants fibroblasts, which arecapable of expressing follistatin-3 polypeptides, onto a patient.Generally, fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin) is added. The flasks are then incubated at 37 degreeC. for approximately one week.

[0638] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

[0639] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HINDIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0640] The cDNA encoding follistatin-3 can be amplified using PCRprimers which correspond to the 5′ and 3′ end sequences respectively asset forth in Example 1. Preferably, the 5′ primer contains an EcoRI siteand the 3′ primer includes a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the amplified EcoRI andHindIII fragment are added together, in the presence of T4 DNA ligase.The resulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB11, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector containsproperly inserted follistatin-3.

[0641] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the follistatin-3 gene is then added to the media andthe packaging cells transduced with the vector. The packaging cells nowproduce infectious viral particles containing the follistatin-3 gene(the packaging cells are now referred to as producer cells).

[0642] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether follistatin-3 protein is produced.

[0643] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 13 Gene Therapy Using Endogenous Follistatin-3 Gene

[0644] Another method of gene therapy according to the present inventioninvolves operably associating the endogenous follistatin-3 sequence witha promoter via homologous recombination as described, for example, inU.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International PublicationNo. WO 96/29411, published Sep. 26, 1996; International Publication No.WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad.Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438(1989). This method involves the activation of a gene which is presentin the target cells, but which is not expressed in the cells, or isexpressed at a lower level than desired.

[0645] Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous follistatin-3, flanking the promoter. The targetingsequence will be sufficiently near the 5′ end of follistatin-3 so thepromoter will be operably linked to the endogenous sequence uponhomologous recombination. The promoter and the targeting sequences canbe amplified using PCR. Preferably, the amplified promoter containsdistinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the3′ end of the first targeting sequence contains the same restrictionenzyme site as the 5′ end of the amplified promoter and the 5′ end ofthe second targeting sequence contains the same restriction site as the3′ end of the amplified promoter.

[0646] The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

[0647] In this Example, the polynucleotide constructs are administeredas naked polynucleotides via electroporation. However, thepolynucleotide constructs may also be administered withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, precipitating agents, etc. Such methods of delivery areknown in the art.

[0648] Once the cells are transfected, homologous recombination willtake place which results in the promoter being operably linked to theendogenous follistatin-3 sequence. This results in the expression offollistatin-3 in the cell. Expression may be detected by immunologicalstaining, or any other method known in the art.

[0649] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in DMEM+10% fetal calf serum. Exponentiallygrowing or early stationary phase fibroblasts are trypsinized and rinsedfrom the plastic surface with nutrient medium. An aliquot of the cellsuspension is removed for counting, and the remaining cells aresubjected to centrifugation. The supernatant is aspirated and the pelletis resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3,137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

[0650] Plasmid DNA is prepared according to standard techniques. Forexample, to construct a plasmid for targeting to the follistatin-3locus, plasmid pUCI 8 (MBI Fermentas, Amherst, N.Y.) is digested withHindIII. The CMV promoter is amplified by PCR with an XbaI site on the5′ end and a BamHI site on the 3′ end. Two follistatin-3 non-codingsequences are amplified via PCR: one follistatin-3 non-coding sequencefollistatin-3 fragment 1) is amplified with a HindIII site at the 5′ endand an Xba site at the 3′ end; the other follistatin-3 non-codingsequence (follistatin-3 fragment 2) is amplified with a BamHI site atthe 5′ end and a HindIII site at the 3′ end. The CMV promoter andfollistatin-3 fragments are digested with the appropriate enzymes (CMVpromoter-XbaI and BamHI; follistatin-3 fragment 1-XbaI follistatin-3fragment 2-BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC 18plasmid.

[0651] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrodegap (Bio-Rad). The final DNA concentration is generally at least 120μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5×10⁶cells) is then added to the cuvette, and the cell suspension and DNAsolutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[0652] Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 of fresh media and incubated for afurther 16-24 hours.

[0653] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product. Thefibroblasts can then be introduced into a patient as described above.

Example 14 Method of Treatment Using Gene Therapy-in vivo

[0654] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) follistatin-3 sequences into an animal toincrease or decrease the expression of the follistatin-3 polypeptide.The follistatin-3 polynucleotide may be operatively linked to a promoteror any other genetic elements necessary for the expression of thefollistatin-3 polypeptide by the target tissue. Such gene therapy anddelivery techniques and methods are known in the art, see, for example,WO90/11092, WO98/11779; U.S. Pat. No. 5693622, 5705151, 5580859; TabataH. et al. (1997) Cardiovasc. Res. 35(3):470-479, Chao J et al. (1997)Pharmacol. Res. 35(6):517-522, Wolff J. A. (1997) Neuromuscul. Disord.7(5):314-318, Schwartz B. et al. (1996) Gene Ther. 3(5):405-411, TsurumiY. et al. (1996) Circulation 94(12):3281-3290 (incorporated herein byreference).

[0655] The follistatin-3 polynucleotide constructs may be delivered byany method that delivers injectable materials to the cells of an animal,such as, injection into the interstitial space of tissues (heart,muscle, skin, lung, liver, intestine and the like). The follistatin-3polynucleotide constructs can be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

[0656] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the follistatin-3 polynucleotides may also bedelivered in liposome formulations (such as those taught in Felgner P.L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al.(1995) Biol. Cell 85(1):1-7) which can be prepared by methods well knownto those skilled in the art.

[0657] The follistatin-3 polynucleotide vector constructs used in thegene therapy method are preferably constructs that will not integrateinto the host genome nor will they contain sequences that allow forreplication. Any strong promoter known to those skilled in the art canbe used for driving the expression of DNA. Unlike other gene therapiestechniques, one major advantage of introducing naked nucleic acidsequences into target cells is the transitory nature of thepolynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

[0658] The follistatin-3 polynucleotide construct can be delivered tothe interstitial space of tissues within the an animal, including ofmuscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart,lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[0659] For the naked follistatin-3 polynucleotide injection, aneffective dosage amount of DNA or RNA will be in the range of from about0.05 g/kg body weight to about 50 mg/kg body weight. Preferably thedosage will be from about 0.005 mg/kg to about 20 mg/kg and morepreferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as theartisan of ordinary skill will appreciate, this dosage will varyaccording to the tissue site of injection. The appropriate and effectivedosage of nucleic acid sequence can readily be determined by those ofordinary skill in the art and may depend on the condition being treatedand the route of administration. The preferred route of administrationis by the parenteral route of injection into the interstitial space oftissues. However, other parenteral routes may also be used, such as,inhalation of an aerosol formulation particularly for delivery to lungsor bronchial tissues, throat or mucous membranes of the nose. Inaddition, naked follistatin-3 polynucleotide constructs can be deliveredto arteries during angioplasty by the catheter used in the procedure.

[0660] The dose response effects of injected follistatin-3polynucleotide in muscle in vivo is determined as follows. Suitablefollistatin-3 template DNA for production of mRNA coding forfollistatin-3 polypeptide is prepared in accordance with a standardrecombinant DNA methodology. The template DNA, which may be eithercircular or linear, is either used as naked DNA or complexed withliposomes. The quadriceps muscles of mice are then injected with variousamounts of the template DNA.

[0661] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The follistatin-3 template DNA is injected in 0.1ml of carrier in a 1 cc syringe through a 27 gauge needle over oneminute, approximately 0.5 cm from the distal insertion site of themuscle into the knee and about 0.2 cm deep. A suture is placed over theinjection site for future localization, and the skin is closed withstainless steel clips.

[0662] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15urn cross-section of the individual quadriceps muscles ishistochemically stained for follistatin-3 protein expression. A timecourse for follistatin-3 protein expression may be done in a similarfashion except that quadriceps from different mice are harvested atdifferent times. Persistence of follistatin-3 DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using follistatin-3 naked DNA.

Example 15 Follistatin-3 Transgenic Animals

[0663] The follistatin-3 polypeptides can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express polypeptides of the invention in humans, as part ofa gene therapy protocol.

[0664] Any technique known in the art may be used to introduce thetransgene (i.e., polynucleotides of the invention) into animals toproduce the founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection (Paterson etal., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,Biotechnology (NY) 11: 1263-1270 (1993); Wright et al., Biotechnology(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191(1989)); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)),blastocysts or embryos; gene targeting in embryonic stem cells (Thompsonet al., Cell 56:313-321 (1989)); electroporation of cells or embryos(Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of thepolynucleotides of the invention using a gene gun (see, e.g., Ulmer etal., Science 259:1745 (1993); introducing nucleic acid constructs intoembryonic pleuripotent stem cells and-transferring the stem cells backinto the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,Cell 57:717-723 (1989); etc. For a review of such techniques, seeGordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989),which is incorporated by reference herein in its entirety.

[0665] Any technique known in the art may be used to produce transgenicclones containing polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

[0666] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.

[0667] Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous geneare designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous gene. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous gene in only that cell type, byfollowing, for example, the teaching of Gu et al. (Gu et al., Science265:103-106 (1994)). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art. Thecontents of each of the documents recited in this paragraph is hereinincorporated by reference in its entirety.

[0668] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

[0669] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

[0670] Transgenic animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of follistatin-3 polypeptides, studying conditionsand/or disorders associated with aberrant follistatin-3 expression, andin screening for compounds effective in ameliorating such conditionsand/or disorders.

Example 16 Follistatin-3 Knock-Out Animals

[0671] Endogenous follistatin-3 gene expression can also be reduced byinactivating or “knocking out” the follistatin-3 gene and/or itspromoter using targeted homologous recombination. (E.g., see Smithies etal., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512(1987); Thompson et al., Cell 5:313-321 (1989); each of which isincorporated by reference herein in its entirety). For example, amutant, non-functional polynucleotide of the invention (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenouspolynucleotide sequence (either the coding regions or regulatory regionsof the gene) can be used, with or without a selectable marker and/or anegative selectable marker, to transfect cells that express polypeptidesof the invention in vivo. In another embodiment, techniques known in theart are used to generate knockouts in cells that contain, but do notexpress the gene of interest. Insertion of the DNA construct, viatargeted homologous recombination, results in inactivation of thetargeted gene. Such approaches are particularly suited in research andagricultural fields where modifications to embryonic stem cells can beused to generate animal offspring with an inactive targeted gene (e.g.,see Thomas & Capecchi 1987 and Thompson 1989, supra). However thisapproach can be routinely adapted for use in humans provided therecombinant DNA constructs are directly administered or targeted to therequired site in vivo using appropriate viral vectors that will beapparent to those of skill in the art.

[0672] In further embodiments of the invention, cells that aregenetically engineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe follistatin-3 polypeptides. The engineered cells which express andpreferably secrete the polypeptides of the invention can be introducedinto the patient systemically, e.g., in the circulation, orintraperitoneally.

[0673] Alternatively, the cells can be incorporated into a matrix andimplanted in the body, e.g., genetically engineered fibroblasts can beimplanted as part of a skin graft; genetically engineered endothelialcells can be implanted as part of a lymphatic or vascular graft. (See,for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan &Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated byreference herein in its entirety).

[0674] When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

[0675] Knock-out animals of the invention have uses which include, butare not limited to, animal model systems useful in elaborating thebiological function of follistatin-3 polypeptides, studying conditionsand/or disorders associated with aberrant follistatin-3 expression, andin screening for compounds effective in ameliorating such conditionsand/or disorders.

Example 17 Assays Detecting Stimulation or Inhibition of B CellProliferation and Differentiation.

[0676] Generation of functional humoral immune responses requires bothsoluble and cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

[0677] One of the best studied classes of B-cell co-stimulatory proteinsis the TNF-superfamily. Within this family CD40, CD27, and CD30 alongwith their respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

[0678] In Vitro Assay—Purified follistatin-3 protein, or truncated formsthereof, is assessed for its ability to induce activation,proliferation, differentiation or inhibition and/or death in B-cellpopulations and their precursors. The activity of follistatin-3 proteinon purified human tonsillar B cells, measured qualitatively over thedose range from 0.1 to 10,000 ng/mL, is assessed in a standardB-lymphocyte co-stimulation assay in which purified tonsillar B cellsare cultured in the presence of either formalin-fixed Staphylococcusaureus Cowan I (SAC) or immobilized anti-human IgM antibody as thepriming agent. Second signals such as IL-2 and IL-15 synergize with SACand IgM crosslinking to elicit B cell proliferation as measured bytritiated-thymidine incorporation. Novel synergizing agents can bereadily identified using this assay. The assay involves isolating humantonsillar B cells by magnetic bead (MACS) depletion of CD3-positivecells. The resulting cell population is greater than 95% B cells asassessed by expression of CD45R(B220).

[0679] Various dilutions of each sample are placed into individual wellsof a 96-well plate to which are added 105 B-cells suspended in culturemedium (RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100U/ml penicillin,10 ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of150 ul. Proliferation or inhibition is quantitated by a 20h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

[0680] In Vivo Assay—BALB/c mice are injected (i.p.) twice per day withbuffer only, or 2 mg/Kg of follistatin-3 protein, or truncated formsthereof. Mice receive this treatment for 4 consecutive days, at whichtime they are sacrificed and various tissues and serum collected foranalyses. Comparison of H&E sections from normal and follistatin-3protein-treated spleens identify the results of the activity offollistatin-3 protein on spleen cells, such as the diffusion ofperi-arterial lymphatic sheaths, and/or significant increases in thenucleated cellularity of the red pulp regions, which may indicate theactivation of the differentiation and proliferation of B-cellpopulations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

[0681] Flow cytometric analyses of the spleens from follistatin-3protein-treated mice is used to indicate whether follistatin-3 proteinspecifically increases the proportion of ThB+, CD45R(B220)dull B cellsover that which is observed in control mice.

[0682] Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andfollistatin-3 protein-treated mice.

[0683] The studies described in this example tested activity infollistatin-3 protein. However, one skilled in the art could easilymodify the exemplified studies to test the activity of follistatin-3polynucleotides (e.g., gene therapy), agonists, and/or antagonists offollistatin-3.

Example 18 T Cell Proliferation Assay

[0684] A CD3-induced proliferation assay is performed on PBMCs and ismeasured by the uptake of ³H-thymidine. The assay is performed asfollows. Ninety-six well plates are coated with 100 microliters/well ofmAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1)overnight at 4 C (1 micrograms/ml in 0.05M bicarbonate buffer, pH 9.5),then washed three times with PBS. PBMC are isolated by F/H gradientcentrifugation from human peripheral blood and added to quadruplicatewells (5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS andP/S in the presence of varying concentrations of follistatin-3 protein(total volume 200 microliters). Relevant protein buffer and medium aloneare controls. After 48 hr. culture at 37 C, plates are spun for 2 min.at 1000 rpm and 100 microliters of supernatant is removed and stored −20C for measurement of IL-2 (or other cytokines) if effect onproliferation is observed. Wells are supplemented with 100 microlitersof medium containing 0.5 microCi of ³H-thymidine and cultured at 37 Cfor 18-24 hr. Wells are harvested and incorporation of ³H-thymidine usedas a measure of proliferation. Anti-CD3 alone is the positive controlfor proliferation. IL-2 (100 U/ml) is also used as a control whichenhances proliferation. Control antibody which does not induceproliferation of T cells is used as the negative controls for theeffects of follistatin-3 proteins.

[0685] The studies described in this example tested activity infollistatin-3 protein. However, one skilled in the art could easilymodify the exemplified studies to test the activity of follistatin-3polynucleotides (e.g., gene therapy), agonists, and/or antagonists offollistatin-3.

[0686] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples. Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0687] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

[0688] Further, the Sequence Listing submitted herewith in paper form ishereby incorporated by reference in its entirety. Moreover, each of theSequence Listings submitted with the following applications, in bothcomputer and paper forms, is hereby incorporated by reference in itsentirety: U.S. application Ser. No. 09/617,804, filed Jul. 14, 2000;U.S. Provisional Application Serial No. 60/144,088, filed Jul. 16, 1999;U.S. Provisional Application Serial No. 60/056,248, filed Aug. 29, 1997;U.S. application Ser. No. 09/141,027, filed Aug. 27, 1998; and PCTApplication Serial No. PCT/US98/17710, filed Aug. 27, 1998.

1 22 1 2495 DNA Homo sapiens CDS (19)..(807) mat_peptide (97)..(807)sig_peptide (19)..(96) misc_feature (2429) n equals a, t, g, or c 1gccgtctctg cgttcgcc atg cgt ccc ggg gcg cca ggg cca ctc tgg cct 51 MetArg Pro Gly Ala Pro Gly Pro Leu Trp Pro -25 -20 ctg ccc tgg ggg gcc ctggct tgg gcc gtg ggc ttc gtg agc tcc atg 99 Leu Pro Trp Gly Ala Leu AlaTrp Ala Val Gly Phe Val Ser Ser Met -15 -10 -5 -1 1 yggc tcg ggg aac cccgcg ccc ggt ggt gtt tgc tgg ctc cag cag ggc 147 Gly Ser Gly Asn Pro AlaPro Gly Gly Val Cys Trp Leu Gln Gln Gly 5 10 15 ycag gag gcc acc tgc agcctg gtg ctc cag act gat gtc acc cgg gcc 195 Gln Glu Ala Thr Cys Ser LeuVal Leu Gln Thr Asp Val Thr Arg Ala 20 25 30 ygag tgc tgt gcc tcc ggcaac att gac acc gcc tgg tcc aac ctc acc 243 Glu Cys Cys Ala Ser Gly AsnIle Asp Thr Ala Trp Ser Asn Leu Thr 35 40 45 ycac ccg ggg aac aag atcaac ctc ctc ggc ttc ttg ggc ctt gtc cac 291 His Pro Gly Asn Lys Ile AsnLeu Leu Gly Phe Leu Gly Leu Val His 50 55 60 65 ytgc ctt ccc tgc aaa gattcg tgc gac ggc gtg gag tgc ggc ccg ggc 339 Cys Leu Pro Cys Lys Asp SerCys Asp Gly Val Glu Cys Gly Pro Gly 70 75 80 yaag gcg tgc cgc atg ctgggg ggc cgc ccg cgc tgc gag tgc gcg ccc 387 Lys Ala Cys Arg Met Leu GlyGly Arg Pro Arg Cys Glu Cys Ala Pro 85 90 95 ygac tgc tcg ggg ctc ccggcg cgg ttg cag gtc tgc ggc tca gac ggc 435 Asp Cys Ser Gly Leu Pro AlaArg Leu Gln Val Cys Gly Ser Asp Gly 100 105 110 ygcc acc tac cgc gac gagtgc gag ctg cgc gcc gcg cgc tgc cgc ggc 483 Ala Thr Tyr Arg Asp Glu CysGlu Leu Arg Ala Ala Arg Cys Arg Gly 115 120 125 ycac ccg gac ctg agc gtcatg tac cgg ggc cgc tgc cgc aag tcc tgt 531 His Pro Asp Leu Ser Val MetTyr Arg Gly Arg Cys Arg Lys Ser Cys 130 135 140 145 ygag cac gtg gtg tgcccg cgg cca cag tcg tgc gtc gtg gac cag acg 579 Glu His Val Val Cys ProArg Pro Gln Ser Cys Val Val Asp Gln Thr 150 155 160 yggc agc gcc cac tgcgtg gtg tgt cga gcg gcg ccc tgc cct gtg ccc 627 Gly Ser Ala His Cys ValVal Cys Arg Ala Ala Pro Cys Pro Val Pro 165 170 175 ytcc agc ccc ggc caggag ctt tgc ggc aac aac aac gtc acc tac atc 675 Ser Ser Pro Gly Gln GluLeu Cys Gly Asn Asn Asn Val Thr Tyr Ile 180 185 190 ytcc tcg tgc cac atgcgc cag gcc acc tgc ttc ctg ggc cgc tcc atc 723 Ser Ser Cys His Met ArgGln Ala Thr Cys Phe Leu Gly Arg Ser Ile 195 200 205 yggc gtg cgc cac gcgggc agc tgc gca ggc acc cct gag gag ccg cca 771 Gly Val Arg His Ala GlySer Cys Ala Gly Thr Pro Glu Glu Pro Pro 210 215 220 225 yggt ggt gag tctgca gaa gag gaa gag aac ttc gtg tgagcctgca 817 Gly Gly Glu Ser Ala GluGlu Glu Glu Asn Phe Val 230 235 ggacaggcct gggcctggtg cccgaggccccccatcatcc cctgttattt attgccacag 877 cagagtctaa tttatatgcc acggacactccttagagccc ggattcggac cacttgggga 937 tcccagaacc tccctgacga tatcctggaaggactgagga agggaggcct gggggccggc 997 tggtgggtgg gatagacctg cgttccggacactgagcgcc tgatttaggg cccttctcta 1057 ggatgcccca gcccctaccc taagacctattgccggggag gattccacac ttccgctcct 1117 ttggggataa acctattaat tattgctactatcaagaggg ctgggcattc tctgctggta 1177 attcctgaag aggcatgact gcttttctcagccccaagcc tctagtctgg gtgtgtacgg 1237 agggtctagc ctgggtgtgt acggagggtctagcctgggt gagtacggag ggtctagcct 1297 gggtgagtac ggaggatcta gcctgggtgagtacggagag tctagcctgg gtgtgtatgg 1357 aggatctagc ctgggtgagt atggagggtctagcctgggt gagtatggag ggtctagcct 1417 gggtgtgtat ggagggtcta gcctgggtgagtatggaggg tctagcctgg gtgtgtatgg 1477 agggtctagc ctgggtgagt atggagggtctagcctgggt gtgtacggag ggtctagtct 1537 gagtgcgtgt ggggacctca gaacactgtgaccttagccc agcaagccag gcccttcatg 1597 aaggccaaga aggctgccac cattccctgccagcccaaga actccagctt ccccactgcc 1657 tctgtgtgcc cctttgcgtc ctgtgaaggccattgagaaa tgcccagtgt gccccctggg 1717 aaagggcacg gcctgtgctc ctgacacgggctgtgcttgg ccacagaacc acccagcgtc 1777 tcccctgctg ctgtccacgt cagttcatgaggcaacgtcg cgtggtctca gacgtggagc 1837 agccagcggc agctcagagc agggcactgtgtccggcgga gccaagtcca ctctggggga 1897 gctctggcgg ggaccacggg ccactgctcacccactggcc ccgagggggg tgtagacgcc 1957 aagactcacg catgtgtgac atccggagtcctggagccgg gtgtcccagt ggcaccacta 2017 ggtgcctgct gcctccacag tggggttcacacccagggct ccttggtccc ccacaacctg 2077 ccccggccag gcctgcagac ccagactccagccagacctg cctcacccac caatgcagcc 2137 ggggctggcg acaccagcca ggtgctggtcttgggccagt tctcccacga cggctcaccc 2197 tcccctccat ctgcgttgat gctcagaatcgcctacctgt gcctgcgtgt aaaccacagc 2257 ctcagaccag ctatggggag aggacaacacggaggatatc cagcttcccc ggtctggggt 2317 gaggagtgtg gggagcttgg gcatcctcctccagcctcct ccagccccca ggcagtgcct 2377 tacctgtggt gcccagaaaa gtgcccctaggttggtgggt ctacaggagc cncagccagg 2437 cagcccaccc caccctgggg ccctgcctcaccaaggaaat aaagactcaa agaagcct 2495 2 263 PRT Homo sapiens 2 Met Arg ProGly Ala Pro Gly Pro Leu Trp Pro Leu Pro Trp Gly Ala -25 -20 -15 Leu AlaTrp Ala Val Gly Phe Val Ser Ser Met Gly Ser Gly Asn Pro -10 -5 -1 1 5Ala Pro Gly Gly Val Cys Trp Leu Gln Gln Gly Gln Glu Ala Thr Cys 10 15 20Ser Leu Val Leu Gln Thr Asp Val Thr Arg Ala Glu Cys Cys Ala Ser 25 30 35Gly Asn Ile Asp Thr Ala Trp Ser Asn Leu Thr His Pro Gly Asn Lys 40 45 50Ile Asn Leu Leu Gly Phe Leu Gly Leu Val His Cys Leu Pro Cys Lys 55 60 6570 Asp Ser Cys Asp Gly Val Glu Cys Gly Pro Gly Lys Ala Cys Arg Met 75 8085 Leu Gly Gly Arg Pro Arg Cys Glu Cys Ala Pro Asp Cys Ser Gly Leu 90 95100 Pro Ala Arg Leu Gln Val Cys Gly Ser Asp Gly Ala Thr Tyr Arg Asp 105110 115 Glu Cys Glu Leu Arg Ala Ala Arg Cys Arg Gly His Pro Asp Leu Ser120 125 130 Val Met Tyr Arg Gly Arg Cys Arg Lys Ser Cys Glu His Val ValCys 135 140 145 150 Pro Arg Pro Gln Ser Cys Val Val Asp Gln Thr Gly SerAla His Cys 155 160 165 Val Val Cys Arg Ala Ala Pro Cys Pro Val Pro SerSer Pro Gly Gln 170 175 180 Glu Leu Cys Gly Asn Asn Asn Val Thr Tyr IleSer Ser Cys His Met 185 190 195 Arg Gln Ala Thr Cys Phe Leu Gly Arg SerIle Gly Val Arg His Ala 200 205 210 Gly Ser Cys Ala Gly Thr Pro Glu GluPro Pro Gly Gly Glu Ser Ala 215 220 225 230 Glu Glu Glu Glu Asn Phe Val235 3 317 PRT Homo sapiens 3 Met Val Arg Ala Arg His Gln Pro Gly Gly LeuCys Leu Leu Leu Leu 1 5 10 15 Leu Leu Cys Gln Phe Met Glu Asp Arg SerAla Gln Ala Gly Asn Cys 20 25 30 Trp Leu Arg Gln Ala Lys Asn Gly Arg CysGln Val Leu Tyr Lys Thr 35 40 45 Glu Leu Ser Lys Glu Glu Cys Cys Ser ThrGly Arg Leu Ser Thr Ser 50 55 60 Trp Thr Glu Glu Asp Val Asn Asp Asn ThrLeu Phe Lys Trp Met Ile 65 70 75 80 Phe Asn Gly Gly Ala Pro Asn Cys IlePro Cys Lys Glu Thr Cys Glu 85 90 95 Asn Val Asp Cys Gly Pro Gly Lys LysCys Arg Met Asn Lys Lys Asn 100 105 110 Lys Pro Arg Cys Val Cys Ala ProAsp Cys Ser Asn Ile Thr Trp Lys 115 120 125 Gly Pro Val Cys Gly Leu AspGly Lys Thr Tyr Arg Asn Glu Cys Ala 130 135 140 Leu Leu Lys Ala Arg CysLys Glu Gln Pro Glu Leu Glu Val Gln Tyr 145 150 155 160 Gln Gly Arg CysLys Lys Thr Cys Arg Asp Val Phe Cys Pro Gly Ser 165 170 175 Ser Thr CysVal Val Asp Gln Thr Asn Asn Ala Tyr Cys Val Thr Cys 180 185 190 Asn ArgIle Cys Pro Glu Pro Ala Ser Ser Glu Gln Tyr Leu Cys Gly 195 200 205 AsnAsp Gly Val Thr Tyr Ser Ser Ala Cys His Leu Arg Lys Ala Thr 210 215 220Cys Leu Leu Gly Arg Ser Ile Gly Leu Ala Tyr Glu Gly Lys Cys Ile 225 230235 240 Lys Ala Lys Ser Cys Glu Asp Ile Gln Cys Thr Gly Gly Lys Lys Cys245 250 255 Leu Trp Asp Phe Lys Val Gly Arg Gly Arg Cys Ser Leu Cys AspGlu 260 265 270 Leu Cys Pro Asp Ser Lys Ser Asp Glu Pro Val Cys Ala SerAsp Asn 275 280 285 Ala Thr Tyr Ala Ser Glu Cys Ala Met Lys Glu Ala AlaCys Ser Ser 290 295 300 Gly Val Leu Leu Glu Val Lys His Ser Gly Ser CysAsn 305 310 315 4 508 DNA Homo sapiens misc_feature (377) n equals a, t,g, or c 4 aattcggcac gagtttctca gccccaagcc tctagtctgg gtgtgtacggagggtctagc 60 ctgggtgtgt acggagggtc tagcctgggt gagtacggag ggtctagcctgggtgagtac 120 ggagggtcta gcctgggtga gtacggagag tctagcctgg gtgtgtatggaggatctagc 180 ctgggtgagt atggagggtc tagcctgggt gagtatggag ggtctagcctgggtgtgtat 240 ggagggtcta gcctgggtga gtatggaggg tctagcctgg gtgtgtatggagggtctagc 300 ctgggtgagt atggagggtc tagccttggt gtttacggag ggtctagtctgagttcgttt 360 tggggacctc agaacantnt taacctttag cccagnaanc caggcccttaatgaaggcca 420 gaaggttnca ccattcctnc cctnccaaga antcaatttc nnaatncntnttgtnccctt 480 ttggnccttt aagccattta naatncca 508 5 466 DNA Homo sapiensmisc_feature (415) n equals a, t, g, or c 5 ggcgacggcg tggagtgcggcccgggcaag gcgtgccgca tgctgggggg ccgcccgcgc 60 tgcgagtgcg cgcccgactgctcggggctc ccggcgcggt tgcaggtctg cggctcagac 120 ggcgccacct accgcgacgagtgcgagctg cgcgccgcgc gctgccgcgg ccacccggac 180 ctgagcgtca tgtaccggggccgctgccgc aagtcctgtg agcacgtggt gtgcccgcgg 240 ccacagtcgt gcgtcgtggaccagacgggc agcgcccact gcgtggtgtg tcgaagcggc 300 gccctgccct gtgccctccagccccggcca ggagctttgc ggccaacaac aaagttacct 360 aaatttcttc gtgccaaatgcgccaaggcc aactgcttcc tgggccggtt ccatnnggcg 420 tncgccaagc gggcaantttcgcaagcanc cctgaaggag ccgcca 466 6 337 DNA Homo sapiens misc_feature(33) n equals a, t, g, or c 6 cttgagtgcg tgtggggacc tcagaacactgtnaccttag cccagcaagc caggcccttn 60 atgaaggcca agaaggctgc caccattccctnncagccca agaactccag cttccccact 120 gcctctttnt gcccctttgc ntcctgtgaaggccattgag aaatgcccag tgtgccccct 180 gggaaagggc acggcctgtg ctcctgacacgggctgtgct tggccacaga accacccagc 240 gtctcccctg ctgctgtcca cgtnagttcatgaggcaacg tcgcgtggtc ttcagacgtg 300 ggagcagcca gcggcagctc aggaggcagggcactgt 337 7 298 DNA Homo sapiens misc_feature (5) n equals a, t, g, orc 7 ggcanagccg nctggtgggt gggatagacc tgctttccgg acactgagcg cctgatttag 60ggcccttntn taggaatgcc ccanccccta ccctaagacc tattgccggg naggattcca 120cacttccgct cctttgggga taaacctatt aattattgct actatcaaga gggctggggc 180attctntgct ggtaaattcc tgaagaggca tgactgcttt tttaagcccc aagcctctag 240ttntgggtgt tttacggagg ggtctnagcc tngggttgtn gtacgggngg ggttctta 298 8186 DNA Homo sapiens misc_feature (34) n equals a, t, g, or c 8ccggcggagc aaagtccact ctgggggagc tctngcgggg accacgggcc actgctcacc 60cactggcccc gaggggggtg tagacgccaa gactcacgca tgtttgacat ccggagtcct 120ggagccgngt gtcccagtgg caccactagg tgctnnctgc ctccacagtg gggttcacan 180ccaggg 186 9 308 DNA Homo sapiens misc_feature (3) n equals a, t, g, orc 9 ggnagaggtg acaccagcna ggtnctgtnt tggnccantn ctcccacgan ggctcaccct 60cccctccatc tgctttaatg ctncgaatcg cctacctgtg ccctgcntgt aaaccacagc 120tttcaaacca gctatgggga gaggacaaca cggaggatat tccagcttcc ccggtctggg 180gtgaaggagt gtggggagct tgggncatcc tcctccagtn tcctccagcc cccaggnagt 240gnctttaanc tgtgggttgc ccagaaaagt gncccttagg tttgttgggt tttaaangga 300gctttaan 308 10 407 DNA Homo sapiens misc_feature (290) n equals a, t,g, or c 10 ggcacgagcc tgggtgtgta cggagggtct agtctgagtg cgtgtggggcctcagaacac 60 tgtgacctta gcccagcaag ccaggccttc atgaaggcaa gaaggtgccaccattccctg 120 ccagcccaag actccagttc cccactgcct ctgtgtgccc tttgcgtcctgtgaagccat 180 tgagaaatgc ccatgtgccc ctgggaaagg gcacggctgt gtcctgacagggtgtgtttg 240 cacagaccac caggtttcct gtgtgtcagt attatgagga acgtcggtgnttagagtnga 300 gcagcaggga gttagagcag gatntntccg gggcaagtcc attttggggttttgcggaca 360 gggcatgtta ccattgcccg aggggntaga gcagttagat tntgaan 40711 139 DNA Homo sapiens misc_feature (2) n equals a, t, g, or c 11anccagggnt ncttggtccc ccacaacctt ccccggccag gcctncagac ccagacttca 60gccagacctn ccttaaccac caatgcagcc ggggcttgcg acaanagcag gtgctggtct 120tggggcagtt nttccangg 139 12 25 DNA Homo sapiens 12 tcacgccata tgggctcggggaacc 25 13 41 DNA Homo sapiens 13 catccgggta ccttattaca cgaagttctcttcctcttct g 41 14 40 DNA Homo sapiens 14 catcgcggat ccgccatcatgcgtcccggg gcgccagggc 40 15 38 DNA Homo sapiens 15 catccgggta cctcacacgaagttctcttc ctcttctg 38 16 40 DNA Homo sapiens 16 catcgcggat ccgccaccatgcgtcccggg gcgccagggc 40 17 35 DNA Homo sapiens 17 tcaccgctcg agcacgaagttctcttcctc ttctg 35 18 40 DNA Homo sapiens 18 catcgcggat ccgccaccatgcgtcccggg gcgccagggc 40 19 38 DNA Homo sapiens 19 catccgggta cctcacacgaagttctcttc ctcttctg 38 20 9 PRT Primer_Bind Description of ArtificialSequence hemaglutanin tag 20 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 218 PRT Primer_Bind Description of Artificial Sequence Myc tag 21 Asp TyrLys Asp Asp Asp Asp Lys 1 5 22 10 PRT Primer_Bind Description ofArtificial Sequence Myc tag 22 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 15 10

What is claimed is:
 1. An isolated nucleic acid molecule nucleic acidmolecule comprising a polynucleotide having a nucleotide sequence atleast 95% identical to a sequence selected from the group consisting of:(a) a nucleotide sequence encoding the follistatin-3 polypeptide havingthe complete amino acid sequence in SEQ ID NO:2 (i.e., positions −26 to237 of SEQ ID NO:2); (b) a nucleotide sequence encoding thefollistatin-3 polypeptide having the complete amino acid sequence in SEQID NO:2 excepting the N-terminal methionine (i.e., positions −25 to 237of SEQ ID NO:2); (c) a nucleotide sequence encoding the predicted maturefollistatin-3 polypeptide having the amino acid sequence at positions 1to 237 in SEQ ID NO:2; (d) a nucleotide sequence encoding thefollistatin-3 polypeptide having the complete amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 209199; (e) anucleotide sequence encoding the follistatin-3 polypeptide having thecomplete amino acid sequence excepting the N-terminal methionine encodedby the cDNA clone contained in ATCC Deposit No. 209199; (f) a nucleotidesequence encoding the mature follistatin-3 polypeptide having the aminoacid sequence encoded by the cDNA clone contained in ATCC Deposit No.209199; and (g) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e) or (f) above.
 2. Thenucleic acid molecule of claim 1 wherein said polynucleotide has thecomplete nucleotide sequence in FIGS. 1A, 1B, and 1C (SEQ ID NO:1). 3.The nucleic acid molecule of claim 1 wherein said polynucleotide has thenucleotide sequence in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) encoding thefollistatin-3 polypeptide having the amino acid sequence in positions−26 to 237 of SEQ ID NO:2.
 4. The nucleic acid molecule of claim 1wherein said polynucleotide has the nucleotide sequence in FIGS. 1A, 1B,and 1C (SEQ ID NO:1) encoding the follistatin-3 polypeptide having theamino acid sequence in positions −25 to 237 of SEQ ID NO:2.
 5. Thenucleic acid molecule of claim 1 wherein said polynucleotide has thenucleotide sequence in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) encoding themature follistatin-3 polypeptide having the amino acid sequence fromabout 1 to about 237 in SEQ ID NO:2.
 6. An isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequence atleast 95% identical to a sequence selected from the group consisting of:(a) a nucleotide sequence encoding a polypeptide comprising the aminoacid sequence of residues n-237 of SEQ ID NO:2, where n is an integer inthe range of −26-12; (b) a nucleotide sequence encoding a polypeptidecomprising the amino acid sequence of residues −26-m of SEQ ID NO:2,where m is an integer in the range of −26-m of 207 to 237; (c) anucleotide sequence encoding a polypeptide having the amino acidsequence consisting of residues n-m of SEQ ID NO:2, where n and m areintegers as defined respectively in (a) and (b) above; and (d) anucleotide sequence encoding a polypeptide consisting of a portion ofthe complete follistatin-3 amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 209199 wherein said portion excludes from1 to about 37 amino acids from the amino terminus of said complete aminoacid sequence encoded by the cDNA clone contained in ATCC Deposit No.209199; (e) a nucleotide sequence encoding a polypeptide consisting of aportion of the complete follistatin-3 amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 209199 wherein said portionexcludes from 1 to about 20 amino acids from the carboxy terminus ofsaid complete amino acid sequence encoded by the cDNA clone contained inATCC Deposit No. 209199; and (f) a nucleotide sequence encoding apolypeptide consisting of a portion of the complete follistatin-3 aminoacid sequence encoded by the cDNA clone contained in ATCC Deposit No.209199 wherein said portion include a combination of any of the aminoterminal and carboxy terminal deletions in (d) and (e), above.
 7. Thenucleic acid molecule of claim 1 wherein said polynucleotide has thecomplete nucleotide sequence of the cDNA clone contained in ATCC DepositNo.
 209199. 8. The nucleic acid molecule of claim 1 wherein saidpolynucleotide has the nucleotide sequence encoding the follistatin-3polypeptide having the complete amino acid sequence excepting theN-terminal methionine encoded by the cDNA clone contained in ATCCDeposit No.
 209199. 9. The nucleic acid molecule of claim 1 wherein saidpolynucleotide has the nucleotide sequence encoding the maturefollistatin-3 polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No.
 209199. 10. An isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a polynucleotide having anucleotide sequence identical to a nucleotide sequence in (a), (b), (c),(d), (e), (f), or (g) of claim 1 wherein said polynucleotide whichhybridizes does not hybridize under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues.
 11. An isolated nucleic acid moleculecomprising a polynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a follistatin-3 polypeptide having an aminoacid sequence in (a), (b), (c), (d), (e), or (f) of claim
 1. 12. Theisolated nucleic acid molecule of claim 11, which encodes anepitope-bearing portion of a follistatin-3 polypeptide wherein the aminoacid sequence of said portion is selected from the group of sequences inSEQ ID NO:2 consisting of: about about Leu-14 to about Ala-20, fromabout Ser-46 to about Ile-55, from about Gly-88 to about Pro-97, fromabout Gly-1 13 to about Leu-133, from about Arg-138 to about Glu-146,from about Pro-177 to about Thr-191, and from about Gly-219 to aboutVal-237.
 13. A method for making a recombinant vector comprisinginserting an isolated nucleic acid molecule of claim 1 into a vector.14. A recombinant vector produced by the method of claim
 13. 15. Amethod of making a recombinant host cell comprising introducing therecombinant vector of claim 14 into a host cell.
 16. A recombinant hostcell produced by the method of claim
 15. 17. A recombinant method forproducing a follistatin-3 polypeptide, comprising culturing therecombinant host cell of claim 16 under conditions such that saidpolypeptide is expressed and recovering said polypeptide.
 18. Anisolated follistatin-3 polypeptide comprising an amino acid sequence atleast 95% identical to a sequence selected from the group consisting of:(a) the amino acid sequence of the full-length follistatin-3 polypeptidehaving the complete amino acid sequence shown in SEQ ID NO:2 (i.e.,positions −26 to 237 of SEQ ID NO:2); (b) the amino acid sequence of thefull-length follistatin-3 polypeptide having the complete amino acidsequence shown in SEQ ID NO:2 excepting the N-terminal methionine (i.e.,positions −25 to 237 of SEQ ID NO:2); (c) the amino acid sequence of thepredicted mature follistatin-3 polypeptide having the amino acidsequence at positions 1 to 237 in SEQ 1) NO:2; (d) the amino acidsequence of the full-length follistatin-3 polypeptide having thecomplete amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 209199; (e) the amino acid sequence of the full-lengthfollistatin-3 polypeptide having the complete amino acid sequenceexcepting the N-terminal methionine encoded by the cDNA clone containedin ATCC Deposit No. 209199; and (f) the amino acid sequence of themature follistatin-3 polypeptide having the amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No.
 209199. 19. An isolatedpolypeptide comprising an epitope-bearing portion of the follistatin-3protein, wherein said portion is selected from the group consisting of:a polypeptide comprising amino acid residues from about Leu-14 to aboutAla-20 of SEQ ID NO:2; a polypeptide comprising amino acid residues fromabout Ser-46 to about Ile-55 of SEQ ID NO:2; a polypeptide comprisingamino acid residues from about Gly-88 to about Pro-97 of SEQ ID NO:2; apolypeptide comprising amino acid residues from about Gly-1 13 to aboutLeu-133 of SEQ ID NO:2; a polypeptide comprising amino acid residuesfrom about Arg-138 to about Glu-146 of SEQ ID NO:2; a polypeptidecomprising amino acid residues from about Pro-177 to about Thr-191 ofSEQ ID NO:2; and a polypeptide comprising amino acid residues from aboutGly-219 to about Val-237 of SEQ ID NO:2.
 20. An isolated antibody thatbinds specifically to a follistatin-3 polypeptide of claim
 18. 21. Anisolated nucleic acid molecule comprising a polynucleotide having asequence at least 95% identical to a sequence selected from the groupconsisting of: (a) the nucleotide sequence of SEQ ID NO:4; (b) thenucleotide sequence of SEQ ID NO:5; (c) the nucleotide sequence of SEQID NO:6; (d) the nucleotide sequence of SEQ ID NO:7; (e) the nucleotidesequence of SEQ ID NO:8; (f) the nucleotide sequence of SEQ ID NO:9; (g)the nucleotide sequence of SEQ ID NO:10; (h) the nucleotide sequence ofSEQ ID NO:11; (i) the nucleotide sequence of a portion of the sequenceshown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) wherein said portioncomprises at least 50 contiguous nucleotides from nucleotide 1 to 500;and (j) the nucleotide sequence of a portion of the sequence shown inFIGS. 1A, 1B, and 1C (SEQ ID NO:1) wherein said portion consists ofnucleotides 100-500, 200-500, 300-500, 400-500, 100-400, 200-400,300-400, 100-300, 200-300, 100-200, 100-2495, 250-2495, 500-2495,1000-2495, 1500-2495, 2000-2495, 100-2000, 250-2000, 500-2000,1000-2000, 1500-2000, 100-1500, 250-1500, 500-1500, 1000-1500, 100-1000,250-1000, and 500-1000 of SEQ ID NO:1; and (k) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a) through (j)above.